This document discusses cloning vectors and techniques. It explains that cloning vectors require a cloning site where new DNA fragments can be inserted. Vectors are improved by adding multiple cloning sites containing different restriction enzyme recognition sites to allow for multiple insertions. DNA fragments can be inserted into vectors using restriction enzymes or linkers/adaptors. Vectors also require selectable markers like antibiotic resistance genes to identify transformed bacterial cells.
Preparatiion of competent cells & Transformation Practical Sabahat Ali
Competence is ability of bacteria to take up foreign DNA.
Transformation is alteration of genetic material resulting from uptake of exogenous genetic material from surrounding environment through cell membrane .
published a DNA sequencing method in 1977 based on chemical modification of DNA and subsequent cleavage at specific bases. Also known as chemical sequencing, this method allowed purified samples of double-stranded DNA to be used without further cloning.
Maxam-Gilbert sequencing requires radioactive labeling at one 5' end of the DNA and purification of the DNA fragment to be sequenced. Chemical treatment then generates breaks at a small proportion of one or two of the four nucleotide bases in each of four reactions (G, A+G, C, C+T). The concentration of the modifying chemicals is controlled to introduce on average one modification per DNA molecule. Thus a series of labeled fragments is generated, from the radiolabeled end to the first "cut" site in each molecule. The fragments in the four reactions are electrophoresed side by side in denaturing acrylamide gels for size separation. To visualize the fragments, the gel is exposed to X-ray film for autoradiography, yielding a series of dark bands each corresponding to a radiolabeled DNA fragment, from which the sequence may be inferred.
Preparatiion of competent cells & Transformation Practical Sabahat Ali
Competence is ability of bacteria to take up foreign DNA.
Transformation is alteration of genetic material resulting from uptake of exogenous genetic material from surrounding environment through cell membrane .
published a DNA sequencing method in 1977 based on chemical modification of DNA and subsequent cleavage at specific bases. Also known as chemical sequencing, this method allowed purified samples of double-stranded DNA to be used without further cloning.
Maxam-Gilbert sequencing requires radioactive labeling at one 5' end of the DNA and purification of the DNA fragment to be sequenced. Chemical treatment then generates breaks at a small proportion of one or two of the four nucleotide bases in each of four reactions (G, A+G, C, C+T). The concentration of the modifying chemicals is controlled to introduce on average one modification per DNA molecule. Thus a series of labeled fragments is generated, from the radiolabeled end to the first "cut" site in each molecule. The fragments in the four reactions are electrophoresed side by side in denaturing acrylamide gels for size separation. To visualize the fragments, the gel is exposed to X-ray film for autoradiography, yielding a series of dark bands each corresponding to a radiolabeled DNA fragment, from which the sequence may be inferred.
This presentation provides an overview of What is a transposon,different types of transposons, their mechanism of action, examples for each type of transposons, changes caused due to insertion of transposon into the target gene and applications of Transposons. They are controlling factors in gene expression. Jumping genes is a special area of interest in Genetic research.
This is technique used widely for protein separation from a mixture and is very easy and less costly method. Slides cover all essential points about EMSA and it is quite interesting to know that how it detect and separate different proteins and their mobility shift assay.
This presentation provides an overview of What is a transposon,different types of transposons, their mechanism of action, examples for each type of transposons, changes caused due to insertion of transposon into the target gene and applications of Transposons. They are controlling factors in gene expression. Jumping genes is a special area of interest in Genetic research.
This is technique used widely for protein separation from a mixture and is very easy and less costly method. Slides cover all essential points about EMSA and it is quite interesting to know that how it detect and separate different proteins and their mobility shift assay.
Over the past few decades, molecular biologists have developed a number of techniques that can be used to build the structure of the target DNA seed sequence more quickly so as to simplify and standardize the cloning process.
Now a day's these technique is tremendously use for in lab by using foreign Dna to to producing insulin in bacteria , plant with high yielding capacity by using Gene from another species
pBluescript is an example of a combination between plasmids and phages (phagemids).
Phagemids represent a hybrid type of class of vectors that serve to produce single-stranded DNA.
This pdf is about the DNA Libraries / Genomic DNA vs cDNA.
For more details visit on YouTube; @SELF-EXPLANATORY; https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos Thanks...!
This presentation includes basic of PCOS their pathology and treatment and also Ayurveda correlation of PCOS and Ayurvedic line of treatment mentioned in classics.
MATATAG CURRICULUM: ASSESSING THE READINESS OF ELEM. PUBLIC SCHOOL TEACHERS I...NelTorrente
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A Strategic Approach: GenAI in EducationPeter Windle
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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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Let’s explore the intersection of technology and equity in the final session of our DEI series. Discover how AI tools, like ChatGPT, can be used to support and enhance your nonprofit's DEI initiatives. Participants will gain insights into practical AI applications and get tips for leveraging technology to advance their DEI goals.
Biological screening of herbal drugs: Introduction and Need for
Phyto-Pharmacological Screening, New Strategies for evaluating
Natural Products, In vitro evaluation techniques for Antioxidants, Antimicrobial and Anticancer drugs. In vivo evaluation techniques
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June 3, 2024 Anti-Semitism Letter Sent to MIT President Kornbluth and MIT Cor...Levi Shapiro
Letter from the Congress of the United States regarding Anti-Semitism sent June 3rd to MIT President Sally Kornbluth, MIT Corp Chair, Mark Gorenberg
Dear Dr. Kornbluth and Mr. Gorenberg,
The US House of Representatives is deeply concerned by ongoing and pervasive acts of antisemitic
harassment and intimidation at the Massachusetts Institute of Technology (MIT). Failing to act decisively to ensure a safe learning environment for all students would be a grave dereliction of your responsibilities as President of MIT and Chair of the MIT Corporation.
This Congress will not stand idly by and allow an environment hostile to Jewish students to persist. The House believes that your institution is in violation of Title VI of the Civil Rights Act, and the inability or
unwillingness to rectify this violation through action requires accountability.
Postsecondary education is a unique opportunity for students to learn and have their ideas and beliefs challenged. However, universities receiving hundreds of millions of federal funds annually have denied
students that opportunity and have been hijacked to become venues for the promotion of terrorism, antisemitic harassment and intimidation, unlawful encampments, and in some cases, assaults and riots.
The House of Representatives will not countenance the use of federal funds to indoctrinate students into hateful, antisemitic, anti-American supporters of terrorism. Investigations into campus antisemitism by the Committee on Education and the Workforce and the Committee on Ways and Means have been expanded into a Congress-wide probe across all relevant jurisdictions to address this national crisis. The undersigned Committees will conduct oversight into the use of federal funds at MIT and its learning environment under authorities granted to each Committee.
• The Committee on Education and the Workforce has been investigating your institution since December 7, 2023. The Committee has broad jurisdiction over postsecondary education, including its compliance with Title VI of the Civil Rights Act, campus safety concerns over disruptions to the learning environment, and the awarding of federal student aid under the Higher Education Act.
• The Committee on Oversight and Accountability is investigating the sources of funding and other support flowing to groups espousing pro-Hamas propaganda and engaged in antisemitic harassment and intimidation of students. The Committee on Oversight and Accountability is the principal oversight committee of the US House of Representatives and has broad authority to investigate “any matter” at “any time” under House Rule X.
• The Committee on Ways and Means has been investigating several universities since November 15, 2023, when the Committee held a hearing entitled From Ivory Towers to Dark Corners: Investigating the Nexus Between Antisemitism, Tax-Exempt Universities, and Terror Financing. The Committee followed the hearing with letters to those institutions on January 10, 202
How to Add Chatter in the odoo 17 ERP ModuleCeline George
In Odoo, the chatter is like a chat tool that helps you work together on records. You can leave notes and track things, making it easier to talk with your team and partners. Inside chatter, all communication history, activity, and changes will be displayed.
Exploiting Artificial Intelligence for Empowering Researchers and Faculty, In...Dr. Vinod Kumar Kanvaria
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Thinking of getting a dog? Be aware that breeds like Pit Bulls, Rottweilers, and German Shepherds can be loyal and dangerous. Proper training and socialization are crucial to preventing aggressive behaviors. Ensure safety by understanding their needs and always supervising interactions. Stay safe, and enjoy your furry friends!
2. 2.9.2. Cloning sites
• The second necessary characteristic of a
vector is a cloning site, which is where the new
DNA fragment will be inserted. This must of
course be located in a region of the plasmid
that is not essential for replication or any
other functions that we need. It will contain a
unique site at which a specific restriction
enzyme will cut, i.e., the enzyme will cut the
plasmid once only.
Pg: 55
3. • If the vector contains only one cloning site, it
would be difficult to insert more than one
fragment. If you insert say a BamHI fragment
into a site on the vector that has been cut with
BamHI, the resulting recombinant plasmid will
have two BamHI sites: one at each end of the
inserted fragment (Figure 2.21). Since the
recombinant plasmid now has two BamHI
sites, it would be difficult to clone further
BamHI fragments into it.
Pg 56
5. • The problem here is that in many cases we do
want to insert several fragments into the same
plasmid.We may want to combine the expression
signals of one gene with the coding region of
another, or we may want to insert additional
markers that can be used to identify the presence
of the plasmid. The best way around this problem
is to create a multiple cloning site (MCS), i.e., a
short DNA region that contains recognition sites
for a number of different enzymes.
• Insertion of a fragment into the BamHI site, as in
Figure 2.23, will still leave a selection of other
sites available for further inserts.
Pg 57
7. Figure 2.23 A recombinant plasmid formed using the cloning vector pUC18. The lacZ
gene has been disrupted by insertion of a DNA fragment, resulting in white colonies on
X-gal plates.
bla, beta-lactamase (ampicillin resistance) – selective marker; ori, origin of replication;
lacZ’, beta-galactosidase (partial gene); lacI, repressor of lac promoter.
Pg 58
8. Selectable markers
• The need for this arises from the inefficiency of bacterial
transformation. Even with the high-efficiency systems that are
now available for E. coli, the best yield available, using native
plasmid DNA, implies that only about 1% of the bacterial cells
actually take up the DNA.
• In practice the yields are likely to be lower than this – and if you
are using a host other than E. coli, many orders of magnitude
lower. Therefore in order to be able to recover the transformed
clones, it is necessary to prevent growth of the non-transformed
cells (i.e., those cells that have not taken up the plasmid).
• The presence of an antibiotic resistance gene on the plasmid
vector means that you can simply plate out the transformation
mix on an agar plate containing the relevant antibiotic, and only
the transformants will be able to grow
Pg 57
10. .
Pg 68
• The activity of the enzyme b-galactosidase is
easily monitored by including in the growth
medium the chromogenic substrate 5-bromo- 4-
chloro-3-indolyl-b-D-galactoside (Xgal). This
compound is colorless but on cleavage releases a
blue indolyl derivative. On solid medium,
colonies that are expressing active b-
galactosidase are blue in color while those
without the activity are white in color. This is
often referred to as blue/white screening. Since
Xgal is not an inducer of b-galactosidase, the
non-substrate inducer isopropyl-b-D-
thiogalactoside (IPTG) is also added to the
medium
11. The phenomenon of a-complementation of b-
galactosidase is widely used in molecular genetics. The
starting-point for a complementation is the M15 mutant
of E. coli. This has a deletion of residues 11–41 in the
lacZ gene and shows no b-galactosidase activity.
Enzyme activity can be restored to the mutant enzyme in
vitro by adding a cyanogen bromide peptide derived
from amino acid residues 3–92. Complementation can
also be shown in vivo. If a plasmid carrying the N-
terminal fragment of the lacZ gene encompassing the
missing region is introduced into the M15 mutant, then
b-galactosidase is produced, as demonstrated by the
production of a blue color on medium containing
Xgal. Pg 68
12. In practice, the plasmid usually carries the lacI gene and
the first 146 codons of the lacZ gene, because in the early
days of genetic engineering this was a convenient fragment
of DNA to manipulate. Since wild-type b-galactosidase
has 1021 amino acids, it is encoded by a gene 3.1 kb in
length. While a gene of this length is easily manipulated in
vitro, there are practical disadvantages to using the whole
gene.
As will be seen later, it is preferable to keep cloning
vectors and their inserts as small as possible. The
phenomenon of a-complementation allows genetic
engineers to take advantage of the lac system without
having to have the entire Z gene on the vector.
Pg 68
13. • Secondly, by treating linearized plasmid vector
DNA with alkaline phosphatase to remove 5′-
terminal phosphate groups, both recircularization
and plasmid dimer formation are prevented (Fig.
3.8). In this case, circularization of the vector can
occur only by insertion of nonphosphatase-
treated foreign DNA which provides one 5′-
terminal phosphate at each join. One nick at each
join remains unligated, but, after transformation
of host bacteria, cellular repair mechanisms
reconstitute the intact duplex.
The enzyme DNA ligase is the key to joining
DNA molecules in vitro
Pg 67, 70
14. Fig. 3.8 Application of alkaline phosphatase treatment to prevent recircularization
of vector plasmid without insertion of foreign DNA. Pg 70
15. • Joining DNA fragments with cohesive ends by
DNA ligase is a relatively efficient process
which has been used extensively to create
artificial recombinants.
• A modification of this procedure depends
upon the ability of T4 DNA ligase to join blunt-
ended DNA molecules
Pg 70
16. • Blunt ligation is most usefully applied to joining
blunt-ended fragments via linker molecules;
• One such molecule is shown in Fig. 3.9. The
molecule can be ligated to both ends of the
foreign DNA to be cloned, and then treated with
restriction endonuclease to produce a sticky-
ended fragment, which can be incorporated into a
vector molecule that has been cut with the same
restriction endonuclease. Insertion by means of
the linker creates restriction-enzyme target sites at
each end of the foreign DNA and so enables the
foreign DNA to be excised and recovered after
cloning and amplification in the host bacterium.
Pg 70
17. Fig. 3.9 A decameric
linker molecule
containing an EcoRI
target site is joined by T4
DNA ligase to both ends
of blunt ended foreign
DNA. Cohesive ends are
then generated by EcoRI.
This DNA can then be
incorporated into a
vector that has been
treated with the same
restriction
endonuclease.
Pg 71
18. Adaptors and linkers are short doublestranded
DNA molecules that permit different
cleavage sites to be interconnected
• It may be the case that the restriction enzyme used to
generate the cohesive ends in the linker will also cut the
foreign DNA at internal sites. In this situation, the
foreign DNA will be cloned as two or more
subfragments.
• One solution to this problem is to choose another
restriction enzyme, but there may not be a suitable
choice if the foreign DNA is large and has sites for
several restriction enzymes. Another solution is to
methylate internal restriction sites with the appropriate
modification methylase. An example of this is described
in Fig. 6.2 (p. 99). Alternatively, a general solution to the
problem is provided by chemically synthesized adaptor
molecules which have a preformed cohesive end.
Pg 71
19. • Consider a blunt-ended foreign DNA containing
an internal BamHI site (Fig. 3.10), which is to be
cloned in a BamHI-cut vector. The BamHI adaptor
molecule has one blunt end bearing a 5′
phosphate group and a BamHI cohesive end
which is not phosphorylated.
• The adaptor can be ligated to the foreign DNA
ends.
• The foreign DNA plus added adaptors is then
phosphorylated at the 5′ termini and ligated into
the BamHI site of the vector. If the foreign DNA
were to be recovered from the recombinant with
BamHI, it would be obtained in two fragments.
However, the adaptor is designed to contain two
other restriction sites (SmaI, HpaII), which may
enable the foreign DNA to be recovered intact.
Pg 71
20. Fig. 3.10 Use of a BamHI adaptor molecule. A synthetic adaptor molecule is ligated to the
foreign DNA. The adaptor is used in the 5′-hydroxyl form to prevent self polymerization.
The foreign DNA plus ligated adaptors is phosphorylated at the 5′-termini and ligated into
the vector previously cut with BamHI. Pg 71
21. • the only difference between an adaptor and a
linker is that the former has cohesive ends and
the latter has blunt ends.
22. Homopolymer tailing is a general method for
joining DNA molecules that has special uses
• A general method for joining DNA molecules makes use of the
annealing of complementary homopolymer sequences. Thus, by
adding oligo(dA) sequences to the 3′ ends of one population of DNA
molecules and oligo(dT) blocks to the 3′ ends of another population,
the two types of molecule can anneal to form mixed dimeric circles
(Fig. 3.11). An enzyme purified from calf thymus, terminal
deoxynucleotidyltransferase, provides the means by which the
homopolymeric extensions can be synthesized, for if presented with a
single deoxynucleotide triphosphate it will repeatedly add
nucleotides to the 3′ OH termini of a population of DNA molecules.
DNA with exposed 3′ OH groups, such as arise from pretreatment
with phage λ exonuclease or restriction with an enzyme such as PstI,
is a very good substrate for the transferase.
• However, conditions have been found in which the enzyme will
extend even the shielded 3′ OH of 5′ cohesive termini generated by
EcoRI Pg 72
23. Fig. 3.11 Use of calf-thymus terminal deoxynucleotidyltransferase to add complementary
homopolymer tails to two DNA molecules. Pg 72
24. Amplified DNA can be cloned using in
vitro recombination
• Conservative site-specific recombinases are
topoisomerases that catalyze rearrangements
of DNA at specific sequences that are
considerably longer than the cleavage
sequence favored by vaccinia topoisomerase.
These sites can be incorporated into PCR-
amplified fragments by including them at the
5′ end of both of the amplification primers in
exactly the same way as shown in Fig. 3.13 for
restriction sites.
Pg 73
25. Fig. 3.13 Incorporation of extra sequence at the 5′ end of a primer. Two primers have
sequences designed to hybridize at the ends of the target region. Primer 1 has an extra
sequence near its 5′ end which forms a HindIII site (AAGCTT), and primer 2 has an extra
sequence near its 5′ end which forms an EcoRI (GAATTC) site. Each primer has an
additional 5′-terminal sequence of four nucleotides so that the hexanucleotide restriction
sites are placed within the extreme ends of the amplified DNA, and so present good
substrates for endonuclease cleavage.
Pg 74