Biol 390 – Lab 8 Restriction Digest and Gel Electrophoresis
2
Objective
· Digest DNA of pGLO plasmid using restriction endonuclease enzymes.
· Run an agarose gel to separate the DNA fragments.
Background
Restriction enzymes cut DNA at specific sites generating a number of different sized fragments. The size of the fragments will depend on the number of sites the plasmid has and the specific enzyme used. The number of fragments can be predicted by viewing the map of the plasmid
Gel electrophoresis is a means of separating DNA in an electrical field. DNA is negatively charged and so will move to the anode (+). Larger fragments will move slower through the agarose matrix than the smaller molecules. Agarose is a polysaccharide polymer derived from seaweed: it is a purified from agar by removing the agaropectin component. Fragments are visualized using ethidium bromide, which will glow orange when exposed to UV light.
Materials
Restriction digest
· Restriction enzymes: Nhe1 and EcoR1 (New England Biolabs) – (KEEP ON ICE)
· Plasmid prepared in lab 7
· NanoDrop Lite spectrophotometer
· Microfuge tubes – Sterile
· 37 C degree bath – block heater
· Sterile 10ul and 200ul tips
· Bleach bottles for cleaning bench
· 10X NE Cut Smart Buffer – comes with enzyme
· Nitrile gloves
· Sterile DI water
· Shaved ice
· Ice block for enzymes
Gel Electrophoresis
· Agarose
· Sterile miliQ Water
· 15 well comb
· 50x TAE buffer
· DNA ladder – diluted in sample buffer (1 KB)
· Gel loading dye
· Gel electrophoresis chamber
· Power supply
· Ethidium bromide
· Gel Sys – visualization system
_______________________________________________
Procedure
Restriction Digest of plasmid DNA
· Safety: Wear nitrile gloves – prevent DNAase from your hands affecting the reaction and protect yourself from ethidium bromide
· Clean the bench with bleach - prevents exogenous enzymes interfering you’re your digests.
· Use the NanoDrop to determine the amount of DNA in your plasmid prep. Use this information to calculate how much sample you need to pipette into the reaction mix.
· Label an Eppendorf tube ‘+’ and another ‘-‘
· Make up a reaction mix in both tubes as follows for one of your plasmid samples
· add 1ug of DNA from your plasmid prep
· 5ul of 10X NE Cut Smart Buffer
· Sterile DI water to make the reaction mix to 50ul
For the + tube
· DNA
x ul
· 10X NE Cut Smart Buffer
5ul
· Nhe1 (add last to + tube)
1ul
· EcoR1 (add last to + tube)
1ul
· Sterile DI water
To make final volume to 50ul
· Add the restriction enzymes last to the + tube ONLY
· Repeat with the other two plasmid samples
For the – tube
· DNA
x ul
· 10X NE Buffer
5ul
· Nhe1
None
· EcoR1
None
· Sterile DI water
To make final volume to 50ul
· Do not add any enzyme to the ‘-‘ tube
· Repeat with the other two plasmid samples
· Mix the tubes by flicking – DO NOT VORTEX
· Give a 5 second spin in the centrifuge to bring the contents to the bottom
· Incu.
- Lab 9 Preparation for Protein PurificationObjectivesGrow tra.docxoswald1horne84988
- Lab 9 Preparation for Protein Purification
Objectives
Grow transformed E. coli in preparation for isolation and separation of green fluorescent protein.
Backgrounds
When E. coli containing the pGLO plasmid is grown in a medium containing arabinose the green fluorescent protein (GFP) is expressed. GFP can then be Isolated and separated on a SDS PAGE gel.
Cultures will be grown at 32C as it is the optimum temperature for folding of the GFP.
Methods
Before the lab
· Use aseptic technique during set up and inoculation
· Select 3 tubes of broth if using 2ml: Label two ara/amp and one amp. If using 5 ml tubes of LB broth use 2 tubes label one ara/amp and one amp.
· Add (9ul /1ml) each of arabinose and ampicillin to two tubes (ara/amp)
· Add (9ul/1ml) of ampicillin to the third tube (amp)
· Thaw one of your frozen culture tubes and mix
· Add 100ul of frozen cultures to each tube
· Place tubes in a rack and incubate in a shaker overnight at 32oC. Be sure to shake vigorously.
During the lab
· WEAR SAFETY GLASSES - Use a UV lamp to view each tube – remark on your observations
· Transfer 2ml of culture from an ara/amp tube to a 2ml microfuge tube and spin for 5 minutes
· Remove the supernatant into a waste beaker by either pouring or using a pipette – be careful not to disturb the pellet.
· Add 250ul of TE buffer to the tubes and mix in the vortex mixer until the pellet is suspended
· Add 50ul of lysozyme to this tube and mix
· The tube will be collected and placed in the freezer for at least 24hr to lyse the cells
· Select the remaining ara/amp and amp tube. Label two microfuge tubes.
· Transfer 600ul of the ara/amp tube to a microfuge tube and 300 ul of the amp tube to the another microfuge tube.
· Spin for 5 minutes and discard the supernatant.
· Freeze these tubes and their pellets
LAB 10 Topic: Protein Purification
Objective
· Isolate and separate green fluorescent protein
Background
· When E.coli containing the pGLO plasmid is grown in a medium containing arabinose the green fluorescent protein (GFP) is expressed. GFP can then be isolated and separated on a SDS PAGE gel.
· The hydrophobic interaction column (HIC) allows the separation of hydrophobic proteins. GFP has several stretches of hydrophobic amino acids: this will enable it to stick to the beads in the HIC.
·
Materials
Lab
· Samples of lysed cells from lab 9
· Eppendorf tubes (sterile)
· Test tube racks
· P-1000 (200-1000 ul) – sterile
· P-200 (20 to 200 ul) – sterile
· Microfuge
· UV light
· HIC column (Biorad)
· Elution tubes
· Binding buffer (4M NH4SO4/TE pH 8)
· Equilibration buffer
· Wash buffer
· TE buffer (elution)
_____________________________________________________________
Lab Procedure
· Wear safety glasses and gloves.
· Remove the Eppendorf tube containing cells from Ara/amp broth and lysozyme from freezer.
· Thaw on the benchtop.
· Centrifuge the tubes for 10 minutes to bring down the bacterial debris.
· While the .
The extraction of DNA involves three main steps that are cell lysis, protein separation, and DNA purification. Cell lysis is usually performed by incubation of cell in buffer containing detergent and protease. Cellular proteins are salted out or phase separated using organic solvents. Finally DNA is isolated and purified either by alcohol precipitation or adsorption with silica and elution.
RNA, DNA Isolation and cDNA synthesis.pptxASJADRAZA10
Isolation, quantification of nucleic acids from wheat and synthesis of cDNA.
Introduction
List of Genotypes
DNA Isolation (CTAB method)
Qualitative check of DNA- Gel electrophoresis
Quantitative test of DNA- Spectrophotometer
Protocol for RNA Isolation
RNA Confirmation
Normalization of RNA
cDNA Synthesis
Protocol for DNA Isolation of plant
50-100mg (2-3) young leaves were collected, then washed with tap water followed by distilled water in petri dish.
Leaves were ground using ethanol sterilized mortar pestle for 15-20 sec, by taking 1mL extraction buffer.
1mL (1000μL) of extraction buffer was again added to collect paste from mortar pestle & then transferred to the 2 mL micro centrifuge tube.
The sample in the tube is incubated at 65°C in water bath for 35-45 mins. (Contents in the tube was mixed by inverting at an interval for 5-10 mins)
The tubes were cooled for 10 minutes in ice.
The sample of equal vol (2mL) was centrifuged @14,000 rpm for 10 mins.
After that the supernatant was transferred to new 2 mL centrifuge tube and equal volume (as of sample) of chloroform: Isoamyl alcohol (24:1) was added.
Then mixed gently for 5-7 mins by inverting the tubes.
Again centrifuged for 10 mins @10,000 rpm
After centrifugation, three layers were observed in the tube.
a) aqueous phase i.e. DNA+RNA
b) protein coagulate
c) organic phase i.e. Chloroform
Again the supernatant (aqueous phase) was collected in 1.5mL tube and equal volume of ice-cold isopropanol was added and stored in -20°C overnight.
Following day, tubes were again centrifuged @10,000rpm for 10 mins.
The supernatant was discarded without disturbing the DNA pellet.
70% ethanol is taken and 0.5mL of it was added to the sample and mixed by tapping for 5 mins.
Again centrifuged @10,000rpm for 10 mins and the supernatant was discarded.
Pellet (DNA Precipitate) was air dried for 10 mins.
Then dissolved in 50μL TE-1X Buffer and the sample was stored at -20°C.
1g of analytical grade Agarose was weighed.
100 mL of autoclaved 1X TBE was added in flask.
Now heated on the oven until the solution becomes transparent.
Solution was allowed to cool down to 60℃.
2 μL of Ethidium Bromide (EtBr) is added in the flask.
Melted agarose gel was poured into the casting tray along with comb.
Any bubble in the gel was removed.
After solidification of gel, comb was removed gently and then running buffer was added in the electrophoretic tank.
Once gel got solidified, it was transferred it into gel tank.
A parafilm was taken and on it 2μL loading dye and 3μL sample was taken, gently mixed with the pipette tip only.
Then the mixture (sample +loading dye) was loaded into the well.
Then electrophoretic unit was run at 90 volt for 50-55 mins.
After that gel was put into the Gel Doc to see the DNA band
(using UV light).
Bright colour band were observed as in the figure.
Few (100-150mg) young leaves were ground into fine powder using liquid Nitrogen.
The main purpose of these slides is to convey information to the Professors, Lecturers, and Students. These slides contain authentic information about this topic which is mentioned in that.
- Lab 9 Preparation for Protein PurificationObjectivesGrow tra.docxoswald1horne84988
- Lab 9 Preparation for Protein Purification
Objectives
Grow transformed E. coli in preparation for isolation and separation of green fluorescent protein.
Backgrounds
When E. coli containing the pGLO plasmid is grown in a medium containing arabinose the green fluorescent protein (GFP) is expressed. GFP can then be Isolated and separated on a SDS PAGE gel.
Cultures will be grown at 32C as it is the optimum temperature for folding of the GFP.
Methods
Before the lab
· Use aseptic technique during set up and inoculation
· Select 3 tubes of broth if using 2ml: Label two ara/amp and one amp. If using 5 ml tubes of LB broth use 2 tubes label one ara/amp and one amp.
· Add (9ul /1ml) each of arabinose and ampicillin to two tubes (ara/amp)
· Add (9ul/1ml) of ampicillin to the third tube (amp)
· Thaw one of your frozen culture tubes and mix
· Add 100ul of frozen cultures to each tube
· Place tubes in a rack and incubate in a shaker overnight at 32oC. Be sure to shake vigorously.
During the lab
· WEAR SAFETY GLASSES - Use a UV lamp to view each tube – remark on your observations
· Transfer 2ml of culture from an ara/amp tube to a 2ml microfuge tube and spin for 5 minutes
· Remove the supernatant into a waste beaker by either pouring or using a pipette – be careful not to disturb the pellet.
· Add 250ul of TE buffer to the tubes and mix in the vortex mixer until the pellet is suspended
· Add 50ul of lysozyme to this tube and mix
· The tube will be collected and placed in the freezer for at least 24hr to lyse the cells
· Select the remaining ara/amp and amp tube. Label two microfuge tubes.
· Transfer 600ul of the ara/amp tube to a microfuge tube and 300 ul of the amp tube to the another microfuge tube.
· Spin for 5 minutes and discard the supernatant.
· Freeze these tubes and their pellets
LAB 10 Topic: Protein Purification
Objective
· Isolate and separate green fluorescent protein
Background
· When E.coli containing the pGLO plasmid is grown in a medium containing arabinose the green fluorescent protein (GFP) is expressed. GFP can then be isolated and separated on a SDS PAGE gel.
· The hydrophobic interaction column (HIC) allows the separation of hydrophobic proteins. GFP has several stretches of hydrophobic amino acids: this will enable it to stick to the beads in the HIC.
·
Materials
Lab
· Samples of lysed cells from lab 9
· Eppendorf tubes (sterile)
· Test tube racks
· P-1000 (200-1000 ul) – sterile
· P-200 (20 to 200 ul) – sterile
· Microfuge
· UV light
· HIC column (Biorad)
· Elution tubes
· Binding buffer (4M NH4SO4/TE pH 8)
· Equilibration buffer
· Wash buffer
· TE buffer (elution)
_____________________________________________________________
Lab Procedure
· Wear safety glasses and gloves.
· Remove the Eppendorf tube containing cells from Ara/amp broth and lysozyme from freezer.
· Thaw on the benchtop.
· Centrifuge the tubes for 10 minutes to bring down the bacterial debris.
· While the .
The extraction of DNA involves three main steps that are cell lysis, protein separation, and DNA purification. Cell lysis is usually performed by incubation of cell in buffer containing detergent and protease. Cellular proteins are salted out or phase separated using organic solvents. Finally DNA is isolated and purified either by alcohol precipitation or adsorption with silica and elution.
RNA, DNA Isolation and cDNA synthesis.pptxASJADRAZA10
Isolation, quantification of nucleic acids from wheat and synthesis of cDNA.
Introduction
List of Genotypes
DNA Isolation (CTAB method)
Qualitative check of DNA- Gel electrophoresis
Quantitative test of DNA- Spectrophotometer
Protocol for RNA Isolation
RNA Confirmation
Normalization of RNA
cDNA Synthesis
Protocol for DNA Isolation of plant
50-100mg (2-3) young leaves were collected, then washed with tap water followed by distilled water in petri dish.
Leaves were ground using ethanol sterilized mortar pestle for 15-20 sec, by taking 1mL extraction buffer.
1mL (1000μL) of extraction buffer was again added to collect paste from mortar pestle & then transferred to the 2 mL micro centrifuge tube.
The sample in the tube is incubated at 65°C in water bath for 35-45 mins. (Contents in the tube was mixed by inverting at an interval for 5-10 mins)
The tubes were cooled for 10 minutes in ice.
The sample of equal vol (2mL) was centrifuged @14,000 rpm for 10 mins.
After that the supernatant was transferred to new 2 mL centrifuge tube and equal volume (as of sample) of chloroform: Isoamyl alcohol (24:1) was added.
Then mixed gently for 5-7 mins by inverting the tubes.
Again centrifuged for 10 mins @10,000 rpm
After centrifugation, three layers were observed in the tube.
a) aqueous phase i.e. DNA+RNA
b) protein coagulate
c) organic phase i.e. Chloroform
Again the supernatant (aqueous phase) was collected in 1.5mL tube and equal volume of ice-cold isopropanol was added and stored in -20°C overnight.
Following day, tubes were again centrifuged @10,000rpm for 10 mins.
The supernatant was discarded without disturbing the DNA pellet.
70% ethanol is taken and 0.5mL of it was added to the sample and mixed by tapping for 5 mins.
Again centrifuged @10,000rpm for 10 mins and the supernatant was discarded.
Pellet (DNA Precipitate) was air dried for 10 mins.
Then dissolved in 50μL TE-1X Buffer and the sample was stored at -20°C.
1g of analytical grade Agarose was weighed.
100 mL of autoclaved 1X TBE was added in flask.
Now heated on the oven until the solution becomes transparent.
Solution was allowed to cool down to 60℃.
2 μL of Ethidium Bromide (EtBr) is added in the flask.
Melted agarose gel was poured into the casting tray along with comb.
Any bubble in the gel was removed.
After solidification of gel, comb was removed gently and then running buffer was added in the electrophoretic tank.
Once gel got solidified, it was transferred it into gel tank.
A parafilm was taken and on it 2μL loading dye and 3μL sample was taken, gently mixed with the pipette tip only.
Then the mixture (sample +loading dye) was loaded into the well.
Then electrophoretic unit was run at 90 volt for 50-55 mins.
After that gel was put into the Gel Doc to see the DNA band
(using UV light).
Bright colour band were observed as in the figure.
Few (100-150mg) young leaves were ground into fine powder using liquid Nitrogen.
The main purpose of these slides is to convey information to the Professors, Lecturers, and Students. These slides contain authentic information about this topic which is mentioned in that.
page - 2 of 7 Analytical Methods Lab Class Intr.docxaryan532920
page - 2 of 7
Analytical Methods Lab Class
Introduction
The purpose of this lab session is to give you hands-on experience of the polymerase
chain reaction (PCR). For this, you will be given five different DNA plasmids, four
containing different truncated cDNA sequences and the fifth containing the full-length
cDNA for the human DNA glycosylase NEIL3. You will prepare the PCR reaction mixes,
run the PCR in a thermocycler and determine the size of the cDNA by agarose gel
electrophoresis.
Nei-like 3 (or endonuclease VIII like 3) is the largest of a family of three proteins found in
mammalian cells. Each acts as a DNA glycosylase, releasing oxidized bases from double-
stranded and single-stranded DNA. However, in addition to the N-terminal Fpg/Nei DNA
glycosylase domain, NEIL3 also has an extended C-terminal tail of unknown function
comprising several zinc finger domains (Liu et al., 2013). In order to study the function of
these C-terminal domains, several truncations to the hNEIL3 cDNA have been made in
our lab. The shortest cDNA (843 bp) contains only the DNA glycosylase domain and has
been shown to have this activity. Subsequent truncated cDNAs contain more and more of
the zinc finger domains at the C-terminus (1044 bp, 1206 bp, 1506 bp and full-length).
The plasmid used in these experiments (pETDuet2) has been designed for the expression
of active NEIL3 protein in bacterial (Escherichia coli) cells. Because the DNA glycosylase
activity of NEIL3 depends on the removal of the N-terminal methionine residue and the
endogenous E. coli enzyme is not active when the penultimate amino acid is valine (as for
NEIL3), the plasmid also codes for a mutated version of the E. coli methionine amino-
peptidase (EcoMapY168A; Liu et al., 2012). Thus, the plasmid is termed a bicistronic
vector as two proteins are expressed from the same plasmid.
PCR primers are single-stranded oligonucleotides that anneal to either end of the DNA
sequence to be amplified. Here, the forward primer anneals to the start of the NEIL3
sequence and the reverse primer delineates each of the four truncated cDNA sequences
and the full length cDNA. Please note that each of the reverse primers also contains a
non-template XhoI restriction site, preceded by CCG, to aid downstream cloning. The
DNA sequences of the primers are given in Table 1.
Please follow the instructions carefully to improve your chances of success!
page - 3 of 7
Table 1. DNA sequences of the hNEIL3 PCR Primers.
Name DNA sequence Tm
hNEIL3 Forward ATG GTG GA A GG A CCA GGC TGT ACT CTG AAT 73.2°C
843-XhoI reverse CCG CTC GAG TTT TTG ACA GTG AGG ACA GAA ATA TGT CAT
TCT GT
72.1°C
1044-XhoI reverse CCG CTC GAG TGA ATC AAT AGG CCT TGA GGT CAA GC 70.7°C
1236-XhoI reverse CCG CTC GAG ATC TAG TAT CTG GTT TTG CTT TGT TTT TCT
TTC CAA AG
71.9°C
1506-XhoI reverse CCG CTC GAG AGG ATT TAA GGT ACG AGG GCC ATC TGT 70.4°C
Full-length-XhoI
reverse
CCG CTC ...
The slides tells about the basic techniques performed in biotechnology lab. a initiator should be known with these techniques so that it become easier for the one who wants to see himself in a biotechnology field.
BOOK REVIEWS How to write a book review There are two .docxmoirarandell
BOOK REVIEWS: How to write a book review
There are two approaches to book reviewing:
Descriptive reviews give the essential information about a book. This is done with description and
exposition, by stating the perceived aims and purposes of the author, and by quoting striking passages
from the text.
Critical reviews describe and evaluate the book, in terms of accepted literary and historical standards,
and supports this evaluation with evidence from the text. The following pointers are meant to be
suggestions for writing a critical review.
Basic requirements
To write a critical review, the reviewer must know two things:
Knowing the work under review: This demands not only attempting to understand the author's purpose
and how the component parts of the work contribute to that purpose, but also knowledge of the
author: his/her nationality, time period, other works etc.
Requirements of the genre: This means understanding the art form and how it functions. Without such
context, the reviewer has no historical or literary standard upon which to base an evaluation.
Reviewing essentials
Description of the book. Sufficient description should be given so that the reader will have some
understanding of the author's thoughts. This account is not a summary. It can be woven into the critical
remarks.
Discuss the author. Biographical information should be relevant to the subject of the review and
enhance the reader's understanding of the work under discussion.
Appraise the book. A review must be a considered judgment that includes:
a statement of the reviewer's understanding of the author's purpose
how well the reviewer feels the author's purpose has been achieved
evidence to support the reviewer's judgement of the author' achievement.
While you read:
Read the book with care.
Highlight quotable passages.
Note your impressions as you read.
Allow time to assimilate what you read so that the book can be seen in perspective.
Keep in mind the need for a single impression which must be clear to the reader.
The review outline
A review outline gives you an over-all grasp of the organization of the review, to determine the central
point your review will make, to eliminate inessentials or irrelevancies, and to fill in gaps or omissions.
Examine the notes you have made and eliminate those with no relationship to your central
thesis.
By organizing your discussion topics into groups, aspects of the book will emerge: e.g., theme,
character, structure, etc.
Write down all the major headings of the outline and fill in the subdivisions.
All parts should support your thesis or central point.
First draft
Opening paragraphs set the tone of the paper. Possible introductions usually make a statement about
the:
Thesis
Authorial purpose
Topicality of the work or its significance
Comparison of the work to others by the same author or within the same genre.
Book Review #3- The Spirit Catches You and You Fall Down”Ch.docxmoirarandell
Book Review #3- “The Spirit Catches You and You Fall Down”
Chapters 7-12
Do you believe removing Lia from her parent's care was the right choice for her overall wellbeing? Why or why not?
How did the author find an interpreter that was successful in serving as a cultural broker between herself and the Lees?
How did Jeanine Hilt advocate for the Lee family?
Explain how Neil Ernst and the Lees may have differed culturally in their understanding of the value or perception of the Ernst's’ family vacation.
Give three reasons why many Hmong may have resisted leaving the refugee camp (Ban Vanai) in Thailand.
.
Book required Current Issues and Enduring Questions, by Sylvan Ba.docxmoirarandell
Book required: Current Issues and Enduring Questions, by Sylvan Barnet (Links to an external site.), Hugo Bedau (Links to an external site.), John O'Hara (Links to an external site.) ISBN 1319035477 which should be edition 11
REQUIREMENTS:
· Organize ideas in well-developed, coherent, and stylistically sophisticated analytical essays.
· Evaluate and improve his/her writing process by revising and editing his/her won essays
· Apply logical reasoning to identify and evaluate authors’ use of rhetorical techniques, participate in critical thinking class discussions and activities, and compose clearly organized and effectively argued written analyses of those texts.
· Identify, analyze, and question stated and unstated assumptions of texts and draw meaningful inferences about the intentions of authors in context.
· Discuss a variety of argumentative and analytical assignments and demonstrate the effective use of rhetorical strategies and an awareness of style.
· Use a variety of research skills to expand analysis of a primary source, evaluating and incorporating secondary source materials that encompass the social, historical, and critical aspects that provide context for the argument.
About Myself:
Name: James Greene
Occupation: Senior Logistic Analyst/Lead For NAVSUP Fleet Logistic Center (FLC) San Dirgo In Support of Littoral Combat Ships (LCS)
Major: AA In Business Administration this my last I need to achieve goal. Working toward a BA in Business Management from University Of Redlands.
Retired Navy Veteran retired in Jun 2010
Married all three of my children attend Southwestern
Ordain Pastor
Hobbies:
Live Concerts
Bowling
Movies
Traveling
Book required:
Current Issues and Enduring Questions, by
Sylvan Barnet
(Links to an external
site.)
,
Hugo Bedau
(Links to an external site.)
,
John O'Hara
(Links to an external
site.)
ISBN
1319035477
which should be edition 11
R
EQUIREMENTS
:
·
Organize ideas in well
-
developed, coherent, and stylistically sophisticated analytical essays.
·
Evaluate and improve his/her writing process by revising and editing his/her won essays
·
Apply logical reasoning to identify a
nd evaluate authors’ use of rhetorical techniques,
participate in critical thinking class discussions and activities, and compose clearly organized
and effectively argued written analyses of those texts.
·
Identify, analyze, and question stated and unstated
assumptions of texts and draw meaningful
inferences about the intentions of authors in context.
·
Discuss a variety of argumentative and analytical assignments and demonstrate the effective
use of rhetorical strategies and an awareness of style.
·
Use a variet
y of research skills to expand analysis of a primary source, evaluating and
incorporating secondary source materials that encompass the social, historical, and critical
aspects that provide context for the argument.
About Myself:
Name: James Greene
Occupation: Senior
Logistic Analyst/Lead .
Book Review #1- The Spirit Catches You and You Fall Down”Chapte.docxmoirarandell
Book Review #1- “The Spirit Catches You and You Fall Down”
Chapters 1-3
Explain why Foua Yang’s birthdate may have been different in various locations in the medical charts?
Describe how the history of the Hmong people as discussed in chapter two may have influenced Foua and Nao Kao’s perception of the physicians and nurses who appear to be in charge of their daughter’s care?
How do you think to have an interpreter might have improved the outcomes of Lia’s numerous emergency room visits up to this point?
Discuss the differences in conceptual frameworks that may have led Foua and Nao Kao and the caregivers at Merced County hospital to misunderstand one another during Lia’s admissions?
How may have Foua and Nao Kao experienced cultural pain during the experience of Lia’s birth in the United States?
Assignment File(s)
NM 245 Book Review assignment overview
[MSWord]
Previous
Next
.
Book reportGringo viejo- Carlos FuentesThe written book repo.docxmoirarandell
Book report
Gringo viejo- Carlos Fuentes
The written book report must include the following (5 paragraphs,3-4 pages,
in spanish
). always include a bibliography with the name of the book and the author, publisher, and copyright date.
A. introduction-
name, author of the book and brief background of the author. Also, in the introduction there should be a summary of the storys main idea{theme}, or briefly describe what the book is about
B.Body of the report
- the body of the report is made up of several paragraphs. you can start with a paragraph about the main characters, this may or may not include a physical description of the characters, but it will definitively include a description of their personalities.
c.
Figure out which type of conflict or problem exists in the story, and explain it in another paragraph.
no plagerism, double spaced, in spanish
.
Book reference Kouzes, James M. and Posner, Barry Z. The Leadership.docxmoirarandell
Book reference: Kouzes, James M. and Posner, Barry Z. The Leadership Challenge, 5th Ed. New Jersey: Jossey-Bass Inc., 2012.
Using Kouzes and Posner's theory, complete a personal audit--The Leadership Challenge book.
Answers questions such as:
What is a leader?
What is an effective?
What are some different types of leadership?
What are the Five Practices of Exemplary Leadership?
What are characteristics/traits of a leader? Ex. honest, trustworthy, consistent, inspiring
What kind of leader are you?
Attitudes and styles of past managers or leaders you have had.
What are contemporary leaders known for? (their style/traits)
2-3 pages APA Double spaced 12 font. 1 inch margins
.
BOOK PICTURE I POSTED TOO. Go to the the textbook, study chapt.docxmoirarandell
BOOK PICTURE I POSTED TOO.
Go to the the textbook, study
chapter 8
on the media, and discuss these issues:
1.Planned obsolescence: provide
Examples
that should not be in the book but from your own life experience)
Fig. 8.7 in the textbook: Violence in the media, and video games.
Examples
should from your own life experience,
Media globalization: Examples. Is it good or bad for the cultural values of the countries involved?
China and the Internet censorship: Why China is doing what it is doing?
.
Book ListBecker, Ernest The Denial of D.docxmoirarandell
Book List
Becker, Ernest The Denial of Death
Castaneda, Carlos The Journey to Ixlan
Castaneda, Carlos The Active Side of Infinity
Jung, C.G. Modern Man in Search of a Soul
Moore, Thomas Care of the Soul
May, Rollo The Cry for Myth
Peck, M. Scott The Road Less Traveled
Keen, Sam Inward Bound
Huxley, Adlous The Doors of Perception
Jaynes, Julian The Origin of Consciousness in the
Breakdown of the Bicameral Mind
Storr & Stevens Freud & Jung
Singer, June Boundaries of the Soul
Esters, Clarissa Pinkola Women Who Run With the Wolves
Grof, Stanislav Spiritual Emergency
Jung, C.G. Memories, Dreams, Reflections
Hillman, James We’ve Had a Hundred Years of Psychotherapy
And the World’s Getting Worse
Hesse, Herman Steppenwolf
Chodron, Pema The Places that Scare You
Grof, Christina & Stan The Stormy Search for the Self
Jung, C.G. Flying Saucers
Jung, C.G. Psychology and the Occult
Freud, Sigmund Civilization and its Discontents
M. Scott Peck People of the Lie
Baumeister, Roy Evil: Inside Human Violence and Cruelty
Frankl, Viktor Man’s Search for Meaning
Storr, Anthony The Essential Jung
Strassman, Rick DMT: The Spirit Molecule
Watson, John B. Behaviorism
Freud, Sigmund The Interpretation of Dreams
Stevens, Jay Storming Heaven: LSD and the American
Dream
Fromm, Erich Escape from Freedom
Jung, Carl Answer to Job
Kubler-Ross, Elizabeth Death and Dying
Skinner, B.F. Beyond Freedom and Dignity
Amundsen, Christan Insights From the Secret Teachings of Jesus
Ruiz, Don Miguel The Four Agreements
Moody, Raymond Life After Life
Jonas, Hans The Gnostic Religion
Ellis, Albert
The Myth of Self-Esteem: How
Rational Emotive Behavior Therapy Can
Change Your Life Forever.
May, Rollo The Discovery of Being: Writings
.
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Similar to Biol 390 – Lab 8 Restriction Digest and Gel Electrophoresis .docx
page - 2 of 7 Analytical Methods Lab Class Intr.docxaryan532920
page - 2 of 7
Analytical Methods Lab Class
Introduction
The purpose of this lab session is to give you hands-on experience of the polymerase
chain reaction (PCR). For this, you will be given five different DNA plasmids, four
containing different truncated cDNA sequences and the fifth containing the full-length
cDNA for the human DNA glycosylase NEIL3. You will prepare the PCR reaction mixes,
run the PCR in a thermocycler and determine the size of the cDNA by agarose gel
electrophoresis.
Nei-like 3 (or endonuclease VIII like 3) is the largest of a family of three proteins found in
mammalian cells. Each acts as a DNA glycosylase, releasing oxidized bases from double-
stranded and single-stranded DNA. However, in addition to the N-terminal Fpg/Nei DNA
glycosylase domain, NEIL3 also has an extended C-terminal tail of unknown function
comprising several zinc finger domains (Liu et al., 2013). In order to study the function of
these C-terminal domains, several truncations to the hNEIL3 cDNA have been made in
our lab. The shortest cDNA (843 bp) contains only the DNA glycosylase domain and has
been shown to have this activity. Subsequent truncated cDNAs contain more and more of
the zinc finger domains at the C-terminus (1044 bp, 1206 bp, 1506 bp and full-length).
The plasmid used in these experiments (pETDuet2) has been designed for the expression
of active NEIL3 protein in bacterial (Escherichia coli) cells. Because the DNA glycosylase
activity of NEIL3 depends on the removal of the N-terminal methionine residue and the
endogenous E. coli enzyme is not active when the penultimate amino acid is valine (as for
NEIL3), the plasmid also codes for a mutated version of the E. coli methionine amino-
peptidase (EcoMapY168A; Liu et al., 2012). Thus, the plasmid is termed a bicistronic
vector as two proteins are expressed from the same plasmid.
PCR primers are single-stranded oligonucleotides that anneal to either end of the DNA
sequence to be amplified. Here, the forward primer anneals to the start of the NEIL3
sequence and the reverse primer delineates each of the four truncated cDNA sequences
and the full length cDNA. Please note that each of the reverse primers also contains a
non-template XhoI restriction site, preceded by CCG, to aid downstream cloning. The
DNA sequences of the primers are given in Table 1.
Please follow the instructions carefully to improve your chances of success!
page - 3 of 7
Table 1. DNA sequences of the hNEIL3 PCR Primers.
Name DNA sequence Tm
hNEIL3 Forward ATG GTG GA A GG A CCA GGC TGT ACT CTG AAT 73.2°C
843-XhoI reverse CCG CTC GAG TTT TTG ACA GTG AGG ACA GAA ATA TGT CAT
TCT GT
72.1°C
1044-XhoI reverse CCG CTC GAG TGA ATC AAT AGG CCT TGA GGT CAA GC 70.7°C
1236-XhoI reverse CCG CTC GAG ATC TAG TAT CTG GTT TTG CTT TGT TTT TCT
TTC CAA AG
71.9°C
1506-XhoI reverse CCG CTC GAG AGG ATT TAA GGT ACG AGG GCC ATC TGT 70.4°C
Full-length-XhoI
reverse
CCG CTC ...
The slides tells about the basic techniques performed in biotechnology lab. a initiator should be known with these techniques so that it become easier for the one who wants to see himself in a biotechnology field.
BOOK REVIEWS How to write a book review There are two .docxmoirarandell
BOOK REVIEWS: How to write a book review
There are two approaches to book reviewing:
Descriptive reviews give the essential information about a book. This is done with description and
exposition, by stating the perceived aims and purposes of the author, and by quoting striking passages
from the text.
Critical reviews describe and evaluate the book, in terms of accepted literary and historical standards,
and supports this evaluation with evidence from the text. The following pointers are meant to be
suggestions for writing a critical review.
Basic requirements
To write a critical review, the reviewer must know two things:
Knowing the work under review: This demands not only attempting to understand the author's purpose
and how the component parts of the work contribute to that purpose, but also knowledge of the
author: his/her nationality, time period, other works etc.
Requirements of the genre: This means understanding the art form and how it functions. Without such
context, the reviewer has no historical or literary standard upon which to base an evaluation.
Reviewing essentials
Description of the book. Sufficient description should be given so that the reader will have some
understanding of the author's thoughts. This account is not a summary. It can be woven into the critical
remarks.
Discuss the author. Biographical information should be relevant to the subject of the review and
enhance the reader's understanding of the work under discussion.
Appraise the book. A review must be a considered judgment that includes:
a statement of the reviewer's understanding of the author's purpose
how well the reviewer feels the author's purpose has been achieved
evidence to support the reviewer's judgement of the author' achievement.
While you read:
Read the book with care.
Highlight quotable passages.
Note your impressions as you read.
Allow time to assimilate what you read so that the book can be seen in perspective.
Keep in mind the need for a single impression which must be clear to the reader.
The review outline
A review outline gives you an over-all grasp of the organization of the review, to determine the central
point your review will make, to eliminate inessentials or irrelevancies, and to fill in gaps or omissions.
Examine the notes you have made and eliminate those with no relationship to your central
thesis.
By organizing your discussion topics into groups, aspects of the book will emerge: e.g., theme,
character, structure, etc.
Write down all the major headings of the outline and fill in the subdivisions.
All parts should support your thesis or central point.
First draft
Opening paragraphs set the tone of the paper. Possible introductions usually make a statement about
the:
Thesis
Authorial purpose
Topicality of the work or its significance
Comparison of the work to others by the same author or within the same genre.
Book Review #3- The Spirit Catches You and You Fall Down”Ch.docxmoirarandell
Book Review #3- “The Spirit Catches You and You Fall Down”
Chapters 7-12
Do you believe removing Lia from her parent's care was the right choice for her overall wellbeing? Why or why not?
How did the author find an interpreter that was successful in serving as a cultural broker between herself and the Lees?
How did Jeanine Hilt advocate for the Lee family?
Explain how Neil Ernst and the Lees may have differed culturally in their understanding of the value or perception of the Ernst's’ family vacation.
Give three reasons why many Hmong may have resisted leaving the refugee camp (Ban Vanai) in Thailand.
.
Book required Current Issues and Enduring Questions, by Sylvan Ba.docxmoirarandell
Book required: Current Issues and Enduring Questions, by Sylvan Barnet (Links to an external site.), Hugo Bedau (Links to an external site.), John O'Hara (Links to an external site.) ISBN 1319035477 which should be edition 11
REQUIREMENTS:
· Organize ideas in well-developed, coherent, and stylistically sophisticated analytical essays.
· Evaluate and improve his/her writing process by revising and editing his/her won essays
· Apply logical reasoning to identify and evaluate authors’ use of rhetorical techniques, participate in critical thinking class discussions and activities, and compose clearly organized and effectively argued written analyses of those texts.
· Identify, analyze, and question stated and unstated assumptions of texts and draw meaningful inferences about the intentions of authors in context.
· Discuss a variety of argumentative and analytical assignments and demonstrate the effective use of rhetorical strategies and an awareness of style.
· Use a variety of research skills to expand analysis of a primary source, evaluating and incorporating secondary source materials that encompass the social, historical, and critical aspects that provide context for the argument.
About Myself:
Name: James Greene
Occupation: Senior Logistic Analyst/Lead For NAVSUP Fleet Logistic Center (FLC) San Dirgo In Support of Littoral Combat Ships (LCS)
Major: AA In Business Administration this my last I need to achieve goal. Working toward a BA in Business Management from University Of Redlands.
Retired Navy Veteran retired in Jun 2010
Married all three of my children attend Southwestern
Ordain Pastor
Hobbies:
Live Concerts
Bowling
Movies
Traveling
Book required:
Current Issues and Enduring Questions, by
Sylvan Barnet
(Links to an external
site.)
,
Hugo Bedau
(Links to an external site.)
,
John O'Hara
(Links to an external
site.)
ISBN
1319035477
which should be edition 11
R
EQUIREMENTS
:
·
Organize ideas in well
-
developed, coherent, and stylistically sophisticated analytical essays.
·
Evaluate and improve his/her writing process by revising and editing his/her won essays
·
Apply logical reasoning to identify a
nd evaluate authors’ use of rhetorical techniques,
participate in critical thinking class discussions and activities, and compose clearly organized
and effectively argued written analyses of those texts.
·
Identify, analyze, and question stated and unstated
assumptions of texts and draw meaningful
inferences about the intentions of authors in context.
·
Discuss a variety of argumentative and analytical assignments and demonstrate the effective
use of rhetorical strategies and an awareness of style.
·
Use a variet
y of research skills to expand analysis of a primary source, evaluating and
incorporating secondary source materials that encompass the social, historical, and critical
aspects that provide context for the argument.
About Myself:
Name: James Greene
Occupation: Senior
Logistic Analyst/Lead .
Book Review #1- The Spirit Catches You and You Fall Down”Chapte.docxmoirarandell
Book Review #1- “The Spirit Catches You and You Fall Down”
Chapters 1-3
Explain why Foua Yang’s birthdate may have been different in various locations in the medical charts?
Describe how the history of the Hmong people as discussed in chapter two may have influenced Foua and Nao Kao’s perception of the physicians and nurses who appear to be in charge of their daughter’s care?
How do you think to have an interpreter might have improved the outcomes of Lia’s numerous emergency room visits up to this point?
Discuss the differences in conceptual frameworks that may have led Foua and Nao Kao and the caregivers at Merced County hospital to misunderstand one another during Lia’s admissions?
How may have Foua and Nao Kao experienced cultural pain during the experience of Lia’s birth in the United States?
Assignment File(s)
NM 245 Book Review assignment overview
[MSWord]
Previous
Next
.
Book reportGringo viejo- Carlos FuentesThe written book repo.docxmoirarandell
Book report
Gringo viejo- Carlos Fuentes
The written book report must include the following (5 paragraphs,3-4 pages,
in spanish
). always include a bibliography with the name of the book and the author, publisher, and copyright date.
A. introduction-
name, author of the book and brief background of the author. Also, in the introduction there should be a summary of the storys main idea{theme}, or briefly describe what the book is about
B.Body of the report
- the body of the report is made up of several paragraphs. you can start with a paragraph about the main characters, this may or may not include a physical description of the characters, but it will definitively include a description of their personalities.
c.
Figure out which type of conflict or problem exists in the story, and explain it in another paragraph.
no plagerism, double spaced, in spanish
.
Book reference Kouzes, James M. and Posner, Barry Z. The Leadership.docxmoirarandell
Book reference: Kouzes, James M. and Posner, Barry Z. The Leadership Challenge, 5th Ed. New Jersey: Jossey-Bass Inc., 2012.
Using Kouzes and Posner's theory, complete a personal audit--The Leadership Challenge book.
Answers questions such as:
What is a leader?
What is an effective?
What are some different types of leadership?
What are the Five Practices of Exemplary Leadership?
What are characteristics/traits of a leader? Ex. honest, trustworthy, consistent, inspiring
What kind of leader are you?
Attitudes and styles of past managers or leaders you have had.
What are contemporary leaders known for? (their style/traits)
2-3 pages APA Double spaced 12 font. 1 inch margins
.
BOOK PICTURE I POSTED TOO. Go to the the textbook, study chapt.docxmoirarandell
BOOK PICTURE I POSTED TOO.
Go to the the textbook, study
chapter 8
on the media, and discuss these issues:
1.Planned obsolescence: provide
Examples
that should not be in the book but from your own life experience)
Fig. 8.7 in the textbook: Violence in the media, and video games.
Examples
should from your own life experience,
Media globalization: Examples. Is it good or bad for the cultural values of the countries involved?
China and the Internet censorship: Why China is doing what it is doing?
.
Book ListBecker, Ernest The Denial of D.docxmoirarandell
Book List
Becker, Ernest The Denial of Death
Castaneda, Carlos The Journey to Ixlan
Castaneda, Carlos The Active Side of Infinity
Jung, C.G. Modern Man in Search of a Soul
Moore, Thomas Care of the Soul
May, Rollo The Cry for Myth
Peck, M. Scott The Road Less Traveled
Keen, Sam Inward Bound
Huxley, Adlous The Doors of Perception
Jaynes, Julian The Origin of Consciousness in the
Breakdown of the Bicameral Mind
Storr & Stevens Freud & Jung
Singer, June Boundaries of the Soul
Esters, Clarissa Pinkola Women Who Run With the Wolves
Grof, Stanislav Spiritual Emergency
Jung, C.G. Memories, Dreams, Reflections
Hillman, James We’ve Had a Hundred Years of Psychotherapy
And the World’s Getting Worse
Hesse, Herman Steppenwolf
Chodron, Pema The Places that Scare You
Grof, Christina & Stan The Stormy Search for the Self
Jung, C.G. Flying Saucers
Jung, C.G. Psychology and the Occult
Freud, Sigmund Civilization and its Discontents
M. Scott Peck People of the Lie
Baumeister, Roy Evil: Inside Human Violence and Cruelty
Frankl, Viktor Man’s Search for Meaning
Storr, Anthony The Essential Jung
Strassman, Rick DMT: The Spirit Molecule
Watson, John B. Behaviorism
Freud, Sigmund The Interpretation of Dreams
Stevens, Jay Storming Heaven: LSD and the American
Dream
Fromm, Erich Escape from Freedom
Jung, Carl Answer to Job
Kubler-Ross, Elizabeth Death and Dying
Skinner, B.F. Beyond Freedom and Dignity
Amundsen, Christan Insights From the Secret Teachings of Jesus
Ruiz, Don Miguel The Four Agreements
Moody, Raymond Life After Life
Jonas, Hans The Gnostic Religion
Ellis, Albert
The Myth of Self-Esteem: How
Rational Emotive Behavior Therapy Can
Change Your Life Forever.
May, Rollo The Discovery of Being: Writings
.
Book is Media Literacy. Eighth EditionW.JamesPotte.docxmoirarandell
Book is
Media Literacy. Eighth Edition
W.
James
Potter
University
of
California,
Santa
Barbara
Respond to the following in a minimum of 175 words:
Describe the process of creating meaning.
Provide an example of how you might assign meaning to a media message you have encountered.
.
Book Forensic and Investigative AccountingPlease answer t.docxmoirarandell
Book: Forensic and Investigative Accounting
Please answer the questions listed below and submit in a word document.
Exercise 41.
What are Howard M. Schilit’s seven financial shenanigans?
Exercise 71.
Go to the FBI internet site or search other sources and prepare a report as to the fraudulent activities in these companies. How did the people pull off the fraud?
a.
Quest Communication.
b.
AmeriFunding.
.
Book Criminoloy Second EditionRead Chapter 6. Please submit .docxmoirarandell
Book "Criminoloy Second Edition
"
Read Chapter 6. Please submit your responses to the following questions via the drop box:
1. What is
social disorganization
? How does it contribute to crime? What were Shaw and McKay's findings with regards to the
Concentric Zone
model?
2. Define
anomie. How does this "cause" crime.
3. Briefly explain Robert K. Merton's
Mean/Ends Theory (Modes of Adaptation).
4. According to Robert Agnew, what are the 3 major types of
negative relationships
which cause
strain
?
5. What would
Albert Cohen
say caused crime? What are
middle-class measuring rods
?
6. How do
Sykes and Matza
differ from Cohen in their belief of crime causation?
7. Briefly explain the
violent subculture theory
of Marvin Wolfgang.
.
Book Discussion #2 Ideas(may select 1 or more to respond to).docxmoirarandell
Book Discussion #2 Ideas
(may select 1 or more to respond to) submit to Discussion Drop Box by 3/1 at 11:59 pm
:
1. Write on contrasting Kant's approach to ethics with consequentialism. Which do you think is better, and why?
2. Explain Kant's principle of universalizability and the principle of humanity. Do they ever give conflicting advice? If so, which do you think is a better guide to our moral obligations?
3. Kant claims that humans have a special kind of value not possessed by anything else on earth. How does he justify this claim? What are the implications of this view regarding the moral status of non-human animals? Do you find this view plausible?
4. What gives actions
moral worth
, according to Kant? Compare Kant's view on this subject with the view of the utilitarian. Which view do you think is preferable, and why?
See RUBRIC and Example tabs (Maximum 30% similarity). Submit in Discussion Drop Box. No late assignments.
.
BOOK 1984 MiniProject What makes a human beingOne .docxmoirarandell
BOOK 1984
MiniProject: What makes a human being?
One of the themes of 1984 is human dignity. In Part Two, Winston’s dreams and memories of his
mother lead him to an appreciation of the proles and to the realization that “the proles had stayed
human” (165). In Part Three, O’Brien refers to Winston as “the last man...the guardian of the
human spirit” (270).
Step 1: Write to analyze and explain your perspective on what it means to be human. Your writing
should be 1-2 pages typed and printed. Think about all of the qualities that make a person
“human” according to Winston—qualities that Winston says the Party has taken away and that
Winston has had to “relearn by conscious effort” (165). Consider those qualities in your analysis
and emphasize and/or add the qualities that you feel are most important to being human. Be sure
to reflect the importance of each of the qualities both within the novel as well as importance to the
human experience.
Step 2: Choose from the options below or create your own (must be approved) to present/
illustrate your analysis:
2. Create a “recipe” that contains all of the essential “ingredients” that make up a human being.
3. Write your own lyrics to a song that explains what it means to be human.
4. Reflect key events from Winston or Julia’s point of view (ex. diary, social media account, video).
5. Make a written, audio, video, visual recording of Winston’s diary throughout the novel.
6. Create an interview with one of the characters (ex. News broadcast, talk show).
10. Create your own original ending for the novel.
Conflict Resolution Strategies
Outline
Conflict Resolution Strategies – FH (Cultural Clashes in Workplace)
I. Understanding the conflict
· Identify contributing factors to conflicts in work environment.
· Identify the parties involved in the conflict.
· Approach towards achieving resolution.
II. Goals
· The short-term goal of conflict resolution.
· The long-term goals of conflict resolution.
III. The actual practice of conflict
· Theoretical information which is the description of conflict resolutions that is to be used.
· Inventive practices that show why this initiative is unique in resolving conflict.
· The step by step instructions of resolving conflict in the workplace.
IV. Conclusion
· The guidebook towards achieving conflict resolution.
· Resources necessary for establishing better conflict resolution.
· Contact information for conflict management groups.
GYPSYLOXX™ Conflict resolution Training ManualWelcome to the GLX Team
The GLX mission is to start a movement to inspire the youth to become their own person; to create a distinctive look that is modern, upscale and versatile; as well as doing our best to assure ultimate Customer satisfaction. As a member of the GLX team, you are responsible for creating a friendly work environment by exhibiting the positive traits listed in this manual.
We were very impressed with your experience and/or skill set and we think you w.
Bonds are a vital source of financing to governments and corpora.docxmoirarandell
Bonds are a vital source of financing to governments and corporations of all types. In this discussion forum, you will have the opportunity to discuss possible sources of risks from the investors’ perspective.
For your initial post, assess what you think are the top three biggest risks for investors associated in bond investments, and explain why. Support your claims with references to at least one recent relevant news article from a credible financial media source (i.e., Bloomberg Business Week, Wall Street Journal, Yahoo Finance, etc.)
.
Bond Company adopted the dollar-value LIFO inventory method on Janua.docxmoirarandell
Bond Company adopted the dollar-value LIFO inventory method on January 1, 2013. In applying the LIFO method, Bond uses internal cost indexes and the multiple-pools approach. The following data were available for Inventory Pool No. 3 for the two years following the adoption of LIFO:
Ending Inventory
At Current
At Base
Year
Cost
Year Cost
Cost index
1/1/13
$305,000
$305,000
1.00
12/31/13
334,360
321,500
1.04
12/31/14
441,440
356,000
1.24
Under the dollar-value LIFO method the inventory at December 31, 2014, should be
.
Boley A Negro Town in the American West (1908) The commu.docxmoirarandell
Boley: A Negro Town in the American West (1908)
The community of Boley, in the Creek Nation of Indian Territory, or what is now Oklahoma,
was one of thirty black towns founded in the West after the Civil War and settled by immigrants
from the South and Middle West. Blacks first arrived in Oklahoma as the slaves of Cherokees
and Creeks. The Indians had been displaced from the Carolinas and Georgia during the 1830s
and forced to relocate by foot along the "Trail of Tears" to new lands in Oklahoma. In 1908, a
year after Oklahoma was granted statehood, Booker T. Washington described the town's
development.
The large proportions of the northward and westward movement of the negro population recall
the Kansas exodus of thirty years ago, when within a few months more than forty thousand
helpless and destitute negroes from the country districts of Arkansas and Mississippi poured into
eastern Kansas in search of "better homes, larger opportunities, and kindlier treatment."
It is a striking evidence of the progress made in thirty years that the present northward and
westward movement of the negro people has brought into these new lands, not a helpless and
ignorant horde of black people--but land-seekers and home-builders, men who have come
prepared to build up the country. In the thirty years since the Kansas exodus the southern negroes
have learned to build schools, to establish banks and conduct newspapers. They have recovered
something of the knack for trade that their foreparents in Africa were famous for. They have
learned through their churches and their secret orders the art of corporate and united action. This
experience has enabled them to set up and maintain in a raw western community, numbering
2,500, an orderly and self-respecting government.
In the fall of 1905 I spent a week in the Territories of Oklahoma and Indian Territory. During the
course of my visit I had an opportunity for the first time to see the three races--the negro, the
Indian, and the white man--living side by side, each in sufficient numbers to make their influence
felt in the communities of which they were a part, and in the Territory as a whole. . . .
One cannot escape the impression, in traveling through Indian Territory, that the Indians, who
own practically all the lands, and until recently had the local government largely in their own
hands, are to a very large extent regarded by the white settlers, who are rapidly filling up the
country, as almost a negligible quantity. To such an extent is this true that the Constitution of
Oklahoma, as I understand it, takes no account of the Indians in drawing its distinctions among
the races. For the Constitution there exist only the negro and the white man. The reason seems to
be that the Indians have either receded--"gone back," as the saying in that region is on the
advance of the white race, or they have intermarried with and become absorbed with it. Indeed,
so rapidly has this interma.
BoF Professional Member Exclusive articles & analysis availa.docxmoirarandell
BoF Professional Member Exclusive: articles & analysis available only to you. View the archive.
lg Professional !
CEO TALK
Burberry Stops Destroying Product and Bans Real Fur
A PR backlash enveloped Burberry following the revelation that it destroyed £28.6 million worth of unsold product last year. Now, the company is ending the practice and banning animal fur. In a global exclusive interview, BoF's
Imran Amed sits down with Burberry CEO Marco Gobbetti to decode the thinking behind the move.
BY IMRAN AMED
SEPTEMBER 6, 2018 05:28
ACTION REQUIRED: You are currently missing out on important BoF Professional membership beneNts. Click here to rectify.
LONDON, United Kingdom — Burberry is stopping its longstanding practice of destroying unsold product after a firestorm of negative press and social media posts in July. That month, it emerged that the British brand had destroyed £28.6 million ($36.8 million) worth of product — including clothing, accessories
and perfume — in fiscal 2017/2018. The company has destroyed £105 million ($135 million) of unsold product in the last five years, a practice it has previously disclosed in its annual reports.
Alongside the shift, Burberry is also banning the use of animal fur — including rabbit, fox, mink and Asiatic raccoon, as well as angora — in its runway collections, beginning with new chief creative officer Riccardo Tisci’s highly anticipated debut collection set to be unveiled on September 17 at London Fashion Week.
Existing fur products will be phased out over time, however the brand will continue to sell products made with shearling.
“Modern luxury means being socially and environmentally responsible. This belief is core to us at Burberry and key to our long-term success,” said chief executive Marco Gobbetti in a statement.
But clearly, the negative publicity was a wake-up call for the British luxury behemoth. “We are in the midst of an environmental crisis exacerbated by the fashion industry,” read an open letter to Burberry from second-hand retailer ThredUp, which captured the sentiment of the backlash. “Fashion is now responsible
for 10 percent of global carbon emissions and is projected to drain a quarter of the world’s carbon budget by 2050. We respect the desire to protect your brand image but discounting your product shouldn’t be scarier than setting it on fire.”
Burberry is not the only fashion or luxury brand to have destroyed product. Last November, H&M was reported to have burned unsold products. According to the New York Times, Nike slashes its unsold sneakers. And, Richemont has reportedly destroyed more than £400 million worth of watches from high-end
brands including Cartier and Jaeger-LeCoultre.
Indeed, it is one of the industry’s dirty secrets that brands regularly destroy product to protect their intellectual property from counterfeiters and to limit the diminished brand perception that comes with disposing of excess stock through heavy discounting.
Burberry says its new.
Introduction to AI for Nonprofits with Tapp NetworkTechSoup
Dive into the world of AI! Experts Jon Hill and Tareq Monaur will guide you through AI's role in enhancing nonprofit websites and basic marketing strategies, making it easy to understand and apply.
Macroeconomics- Movie Location
This will be used as part of your Personal Professional Portfolio once graded.
Objective:
Prepare a presentation or a paper using research, basic comparative analysis, data organization and application of economic information. You will make an informed assessment of an economic climate outside of the United States to accomplish an entertainment industry objective.
2024.06.01 Introducing a competency framework for languag learning materials ...Sandy Millin
http://sandymillin.wordpress.com/iateflwebinar2024
Published classroom materials form the basis of syllabuses, drive teacher professional development, and have a potentially huge influence on learners, teachers and education systems. All teachers also create their own materials, whether a few sentences on a blackboard, a highly-structured fully-realised online course, or anything in between. Despite this, the knowledge and skills needed to create effective language learning materials are rarely part of teacher training, and are mostly learnt by trial and error.
Knowledge and skills frameworks, generally called competency frameworks, for ELT teachers, trainers and managers have existed for a few years now. However, until I created one for my MA dissertation, there wasn’t one drawing together what we need to know and do to be able to effectively produce language learning materials.
This webinar will introduce you to my framework, highlighting the key competencies I identified from my research. It will also show how anybody involved in language teaching (any language, not just English!), teacher training, managing schools or developing language learning materials can benefit from using the framework.
Honest Reviews of Tim Han LMA Course Program.pptxtimhan337
Personal development courses are widely available today, with each one promising life-changing outcomes. Tim Han’s Life Mastery Achievers (LMA) Course has drawn a lot of interest. In addition to offering my frank assessment of Success Insider’s LMA Course, this piece examines the course’s effects via a variety of Tim Han LMA course reviews and Success Insider comments.
A Strategic Approach: GenAI in EducationPeter Windle
Artificial Intelligence (AI) technologies such as Generative AI, Image Generators and Large Language Models have had a dramatic impact on teaching, learning and assessment over the past 18 months. The most immediate threat AI posed was to Academic Integrity with Higher Education Institutes (HEIs) focusing their efforts on combating the use of GenAI in assessment. Guidelines were developed for staff and students, policies put in place too. Innovative educators have forged paths in the use of Generative AI for teaching, learning and assessments leading to pockets of transformation springing up across HEIs, often with little or no top-down guidance, support or direction.
This Gasta posits a strategic approach to integrating AI into HEIs to prepare staff, students and the curriculum for an evolving world and workplace. We will highlight the advantages of working with these technologies beyond the realm of teaching, learning and assessment by considering prompt engineering skills, industry impact, curriculum changes, and the need for staff upskilling. In contrast, not engaging strategically with Generative AI poses risks, including falling behind peers, missed opportunities and failing to ensure our graduates remain employable. The rapid evolution of AI technologies necessitates a proactive and strategic approach if we are to remain relevant.
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Biol 390 – Lab 8 Restriction Digest and Gel Electrophoresis .docx
1. Biol 390 – Lab 8 Restriction Digest and Gel Electrophoresis
2
Objective
· Digest DNA of pGLO plasmid using restriction endonuclease
enzymes.
· Run an agarose gel to separate the DNA fragments.
Background
Restriction enzymes cut DNA at specific sites generating a
number of different sized fragments. The size of the fragments
will depend on the number of sites the plasmid has and the
specific enzyme used. The number of fragments can be
predicted by viewing the map of the plasmid
Gel electrophoresis is a means of separating DNA in an
electrical field. DNA is negatively charged and so will move to
the anode (+). Larger fragments will move slower through the
agarose matrix than the smaller molecules. Agarose is a
polysaccharide polymer derived from seaweed: it is a purified
from agar by removing the agaropectin component. Fragments
are visualized using ethidium bromide, which will glow orange
when exposed to UV light.
Materials
2. Restriction digest
· Restriction enzymes: Nhe1 and EcoR1 (New England Biolabs)
– (KEEP ON ICE)
· Plasmid prepared in lab 7
· NanoDrop Lite spectrophotometer
· Microfuge tubes – Sterile
· 37 C degree bath – block heater
· Sterile 10ul and 200ul tips
· Bleach bottles for cleaning bench
· 10X NE Cut Smart Buffer – comes with enzyme
· Nitrile gloves
· Sterile DI water
· Shaved ice
· Ice block for enzymes
Gel Electrophoresis
· Agarose
· Sterile miliQ Water
· 15 well comb
· 50x TAE buffer
· DNA ladder – diluted in sample buffer (1 KB)
· Gel loading dye
· Gel electrophoresis chamber
· Power supply
· Ethidium bromide
· Gel Sys – visualization system
_______________________________________________
Procedure
Restriction Digest of plasmid DNA
· Safety: Wear nitrile gloves – prevent DNAase from your hands
affecting the reaction and protect yourself from ethidium
bromide
· Clean the bench with bleach - prevents exogenous enzymes
interfering you’re your digests.
· Use the NanoDrop to determine the amount of DNA in your
3. plasmid prep. Use this information to calculate how much
sample you need to pipette into the reaction mix.
· Label an Eppendorf tube ‘+’ and another ‘-‘
· Make up a reaction mix in both tubes as follows for one of
your plasmid samples
· add 1ug of DNA from your plasmid prep
· 5ul of 10X NE Cut Smart Buffer
· Sterile DI water to make the reaction mix to 50ul
For the + tube
· DNA
x ul
· 10X NE Cut Smart Buffer
5ul
· Nhe1 (add last to + tube)
1ul
· EcoR1 (add last to + tube)
1ul
· Sterile DI water
To make final volume to 50ul
· Add the restriction enzymes last to the + tube ONLY
· Repeat with the other two plasmid samples
For the – tube
· DNA
x ul
· 10X NE Buffer
5ul
· Nhe1
None
· EcoR1
None
· Sterile DI water
To make final volume to 50ul
· Do not add any enzyme to the ‘-‘ tube
4. · Repeat with the other two plasmid samples
· Mix the tubes by flicking – DO NOT VORTEX
· Give a 5 second spin in the centrifuge to bring the contents to
the bottom
· Incubate for 65 mins in heating block.
· Place the tubes on ice to stop the reaction
Gel Electrophoresis
For one gel
· Block the ends of the gel mold using time tape. Insert the
sample comb for 8 or 15 wells.
· Place the tray in the in the plastic lid box to catch any leaks.
· Weigh out the amount of agarose needed to prepare 50 ml of
1% agarose
· Transfer the agarose to a 250ml Erlenmeyer flask.
· Dilute 1 ml of 50X TAE buffer to 50 ml in a graduated
cylinder and add to the agarose.
· Place a piece of paper towel in the neck of the flask so it
partially blocks the neck.
· Microwave on high for 30 seconds for up to 4 times. USE A
SILICONE HANDGRABBER. Swirl flask gently in between
microwaving. STOP when the agarose is clear and no more agar
powder is visible.
· Cool on bench until it is comfortable to handle.
· Add 0.5ul of ethidium bromide (CAUTION – MUTAGEN) to
the agarose and gently swirl.
· Allow gel to cool so it can be placed against the inside of the
wrist. Running it under cold water helps, but do not let it set.
· Gently pour the agarose into the template mold and let it set
for 10 minutes or more
· Gently remove the comb and the bumpers from the gel.
· Mix 250ml of 1xTAE using your 50X stock
· Place the gel still in the holder in the gel box.
· Pour buffer into the box until it covers the gel.
· Load 10ul of diluted ladder very slowly to the center lane, and
the end lanes
5. · In clean microfuge tubes make up the following.
· For your + and - samples run these in duplicate at different
levels
)
10
2
20
4
· Load the gel as follows:
Lane
Sample vol
Sample
1
10ul
Ladder -1KB
2
10+2 ul
Isolate 1 + enzyme
3
10+2 ul
Isolate 1 - enzyme
4
10+2 ul
Isolate 2 + enzyme
5
10+2 ul
Isolate 2 - enzyme
6
10+2 ul
Isolate 3 + enzyme
7
10+2 ul
Isolate 3 - enzyme
8
6. 10ul
Ladder -1KB
9
20+4 ul
Isolate 1 + enzyme
10
20+4 ul
Isolate 1 - enzyme
11
20+4 ul
Isolate 2 + enzyme
12
20+4 ul
Isolate 2 - enzyme
13
20+4 ul
Isolate 3 + enzyme
14
20+4 ul
Isolate 3 - enzyme
15
10ul
Ladder – 1KB
· Very slowly add each of the + and – digests either side of the
ladder
· Orient the gel so the sample is at the cathode end (black) and
the samples will flow in the direction of the arrow on the side
of the box.
· Place the top on the box and connect the electrodes and turn
the power on.
· Set the voltage to 150 volts and hit run. The display will
show 150 volts and you should see gas coming from the
electrodes.
· Run until the indicator dye is close to the bottom.
· Move the gel to the Gel illuminator and view the bands.
7. Questions
· See the assignment in Canvas
Biol 390-Lab 7 Select transformants and isolate plasmids
4
Objective
· Select transformed cells.
· Freeze cells for future labs.
· Isolate plasmids from transformed cells
Background
p-GLO plasmid
pGLO contains several DNA sequences that enable replication
of the plasmid DNA and expression of the fluorescent trait
(phenotype) in bacteria following transformation. The essential
sequences include the following:
GFP— the jellyfish gene that codes for the production of green
fluorescent protein
8. bla — a gene that encodes the enzyme beta-lactamase, which
breaks down the antibiotic ampicillin. Bacteria containing the
bla gene can be selected by placing ampicillin in the growth
medium.
ori — the origin of pGLO plasmid DNA replication
araC — a gene that encodes the regulatory protein that binds to
the pBAD promoter. Only when arabinose binds to the AraC
protein is the production of GFP switched on.
pBAD promoter — a specific DNA sequence upstream from the
GFP gene, which binds araC-arabinose and promotes RNA
polymerase binding and transcription of the GFP gene.
Multiple cloning site — a region containing restriction sites
(NdeI, HindIII, EcoRI, etc.), sequences that permit the insertion
or deletion of the gene of interest
Materials
For day before
· Three tubes of LB broth / group
· Ampicillin solution (10mg/ml)
· Sterile plastic loops
Day of the lab
· Sterile tips
· Bleach spray bottles
· Nitrile gloves
· 20% glycerol sterile
· Sterile tubes for freezing cultures
· Midisci high speed plasmid mini kit
· Add RNase A to PD1 buffer and store at 4 oC (prepare day
before)
· Check for precipitates in PD2 buffer – if present warm in
37oC bath
· Add 100 ml of absolute ethanol to wash buffer
_______________________________________________
Procedure
Day before
9. · Add ampicillin to LB tubes containing 5 ml of LB broth.
· How much ampicillin do you need to add to make the final
concentration 100ug/ml
· Label LB tubes with your name an number for each colony.
· Inoculate three separate colonies into three different tubes of
LB. Pick three colonies from the LB amp/ara using sterile a
sterile loop for each transfer.
· Incubate tubes at 37oC
Day of lab
· Observe plates from your experiment. Where possible count
the number of colonies. Which colonies glow?
Plate
Observations
+pGLO LB/amp
+pGLO LB/amp/ara
-pGLO LB/amp
- pGLO LB
Freeze cells
· Label 3 sterile Eppendorf tubes with your name and your
colony number.
· Aseptically transfer 0.5 ml of your overnight culture to a
sterile Eppendorf.
· Using a new pipette add 500ul of 20% glycerol
· Freeze at -80oC
High-Speed Plasmid Mini Kit Protocol -Midisci
· Add provided RNase A to the PD1 Buffer and store at 4oC.
(the day before)
· If precipitates have formed in the PD2 Buffer, warm the buffer
in a 37oC water bath, followed by gentle shaking to dissolve.
10. · Add absolute ethanol to the Wash Buffer prior to initial use
(see the bottle label for volume).
· Additional requirements: microcentrifuge tubes.
Step1 Harvesting
· Transfer 1.5 ml of cultured bacterial cells to a microcentrifuge
tube. Microcentrifuge for 1 minute and discard the supernatant.
Step 2 Re-suspension
· Add 200 μl of PD1 Buffer (RNase A added) to the tube and
resuspend the cell pellet by vortex or pipetting.
Step 3 Lysis
· Add 200 μl of PD2 Buffer and mix gently by inverting the
tube 10 times. Do not vortex to avoid shearing the genomic
DNA.
Let stand at room temperature for 2 minutes or until the lysate
is homologous.
Step 4 Neutralization
· Add 300 μl of PD3 Buffer and mix immediately by inverting
the tube 10 times. Do not vortex.
· Microcentrifuge for 3 minutes.
Step 5 DNA Binding
· Place a PD Column in a 2 ml Collection Tube.
· Add the supernatant from Step 4 to the PD Column and
microcentrifuge for 30 seconds.
· Discard the flow-through and place the PD Column back in the
2 ml Collection Tube.
Step 6 Wash
· Add 400 μl of W1 Buffer into the PD Column.
· Microcentrifuge for 30 seconds.
· Discard the flow-through and place the PD Column back in the
2 ml Collection Tube.
Add 600 μl of Wash Buffer (ethanol added) into the PD
Column. Microcentrifuge for 30 seconds.
· Discard the flow through and place the PD Column back in the
2 ml Collection Tube.
Microcentrifuge again for 3 minutes to dry the column matrix.
Step 7 DNA Elution
11. · Transfer the dried PD Column to a new microcentrifuge tube.
· Add 50 μl of Elution Buffer or TE into the center of the
column matrix.
· Let stand for 2 minutes or until the Elution Buffer or TE is
absorbed by the matrix
· Microcentrifuge for 2 minutes to elute the DNA.
· Store at -20oC
Data Analysis
· Explain that in the +pGlo experiments why the colonies that
grew on the LB/amp/ara fluoresced, while those that grew on
LB/amp did not
· Why do colonies of the –Pglo not grow on the LB/amp plate
References
· MIDIsci
· http://shop.midsci.com/protocals/IB47100Protocol.pdf
· Biorad
· http://www.bio-rad.com/en-us/applications-technologies/pglo-
plasmid-map-resources
·
Biol 390-Lab 6 Transformation
12. 2
Objective
· Transform E. coli with the using a plasmid containing Green
Florescent Protein (GFP)
Background
Green fluorescent protein from a jellyfish. This protein has
been engineered into a plasmid. The plasmid also contains an
arabinose promoter that will cause the GFP to be expressed and
a gene that codes for a beta-lactamase; an enzyme that breaks
down ampicillin.
Plasmid and its genes
Materials
For each group
· E. coli starter plate – streaked for single colonies and 24 hours
old
· Agar plates – 1 LB, 2 LB/amp, 1 LB/amp/ara
· Transformation solution – 1 ml color coded
· LB nutrient broth – 1 ml color coded
· Inoculating loops – plastic disposable
· Sterile pipette tips
· Foam tube holder
· Sterile microfuge tubes
· Ice bath
· Stop watch
· Marker
Common work station
· pGLO plasmid
· Heating block – 42oC
13. · Nitrile gloves
______________________________________________
Prelab Preparation
Two days before
· Add 250ul of LB broth to the lyophilized E. coli using a
sterile tip and incubate for 8 to 24 hr at 370C
One day before
· Streak plates for single colonies – incubate 24 to 36 hours.
Each group gets one plate
Day of the lab
· Prepare the pGLO plasmid – transfer 250ul of the
transformation solution into the lyophilized pGLO vial. Mix by
inverting in case there is plasmid DNA attached to the cap.
· Label one tube per group “TR”: aseptically transfer 1000ul to
each of the transformation solution.
· Lab one tube per group “LB”: aseptically transfer 1000ul to
each of the LB nutrient medium.
Procedure
1. Use gloves during this experiment
2. Clean benches with alcohol – be careful
3. Select two sterile microfuge tubes and label
a. +pGLO
b. –pGLO
4. Place in a rack
5. Open tubes and using a separate sterile pipette tip transfer
250ul of the transformation solution (TR) to each tube.
6. Place tubes in foam rack in the crushed ice bath.
a. Note: make sure the tubes are in contact with the ice. You
may have to push theminto the foam.
7. Using a sterile disposable loop transfer 2 to 4 E.coli colonies
from the agar plate to each tube.
a. NOTE – it important to uses between 2 and 4, 1 to 1.5 mm
colonies to maximize the transformation efficiency.
8. Spin the loop in the transformation solution until all the
E.coli have been suspended
14. a. Note there should be no floating chunks.
9. Use a new loop to inoculate the other tube in the same way
using other colonies from the plate.
10. Transfer a 10ul aliquot of the pGLO plasmid to the +pGLO
tube using a sterile tip and an automatic pipette.
a. NOTE – do not add the plasmid to the –pGLO tube
11. Incubate the tubes on ice for 10 mins once the plasmid has
been added.
12. While the tubes are sitting in the ice label your four plates
with your name.
a. +pGLO LB/amp
b. +pGLO LB/amp/ara/
c. -pGLO LB/amp
d. –pGLO LB
13. Transfer the tubes into the heating block set at 42oC for
exactly 50 seconds. Note – timing is critical
14. After exactly 50 seconds quickly transfer the tubes to ice for
2 minutes.
15. Place the tube back in the microfuge tube rack and add
250ul of LB broth to one tube using a sterile pipette tip. Add
250 ul of broth to the other tube using a new sterile pipette tip.
16. Incubate the tubes for 10 min at room temperature.
17. Gently flick the closed tubes with your finger to mix.
a. Note its important to make sure the tubes are mixed. Pipette
100ul from each tubes as follows:
b. +pGlO tube to:
i. +pGLO LB/amp
ii. +pGLO LB/amp/ara
c. – pGlO tube to:
i. –pGLO LB/amp
ii. –pGLO LB
18. Using a sterile loop spread the suspension gently around the
entire surface of the plate. Note this requires very little
downward pressure.
19. Turn plates upside down and incubate for 24 hours at 37oC
15. Biol 390-Lab 5 Preparation for Transformation
2
Objective
· Prepare materials for transformation
· Prepare TAE buffer for electrophoresis in a later lab.
Background
Transformation of bacterial with plasmids is a fundamental
technique in genetic engineering. Cells can be made competent
to take up plasmids by treatment with divalent cations and heat.
Transformed cells will contain a plasmid that contains a gene
that confers resistance to the antibiotic, ampicillin. Therefore,
transformed cells can be selected by growth on LB/amp plates.
Growth on LB/ara /amp will cause the product to be expressed
as the plasmid contains an arabinose promotor
Materials
· LB broth
· LB agar
· Petri dishes
16. Needed when plates are poured
· Ampicillin (amp) solution sterile – 10 mg/ml stock
· Arabinose (ara) solution sterile – 200 mg/ml stock
For TAE (Tris –acetic acid-EDTA) buffer
· Tris base
· Glacial Acetic acid
· EDTA
_______________________________________________
Procedure
Make LB agar
You will need per group
· 1 LB plates
· 2 LB/ amp plates
· 1 LB /amp/ara
How much volume each will you need for the whole class?
How much does a petri dish hold?
Make up separate bottles of LB agar for each set of the plates
Formula for Luria broth.
· Luria broth – 20g/l
· Agar – 15g/l
Heat the broth agar mixture until the agar melts (about 95oC).
Your bottles will be autoclaved at 121oC for 20 minutes and
stored until 2 days before.
As arabinose or ampicillin can not be autoclaved, sterile
solutions of these will be added to the agar after it is re-melted
2 days before the experiment.
TAE Buffer
You need to prepare 200ml of a 50X stock of TAE buffer to use
for gel electrophoresis
Recipe for one liter of 50X stock ‘
· Dissolve 242g Tris base in water
· add 57.1mL glacial acetic acid,
· add 18.6g EDTA solution
· bring the final volume up to 1 liter.
17. This stock solution will be diluted 50:1 with water to make a
1X working solution. The 1X solution will contain 0.4mM Tris,
20mM acetic acid, and 1mM EDTA.