molcular biology techniques- ravi ranjan lb-
contents- basic molecular biology techniques - DNA and RNA isolation from plant sample, nanodrop technique, pcr and cloning.
methods of isolation and extraction of RNA by using different source such as plant tissues, bacterial culture, etc. Ribonucleic acid can be isolated from plant tissue for the purpose of:
– mRNA isolation
– In vitro translation
– Northern analysis
– cDNA library construction
Rigorous ribonuclease free environment is to be maintained
All glasswares, plasticwares and reagents made RNAse free (using 0.01% DEPC)
Next day, DEPC is inactivated by autoclaving for 30 min
Total RNA is isolated and separated from DNA and protein after extraction with a solution called as Trizol. Trizol is an acidic solution containing guanidinium thiocyanate (GITC), phenol and chloroform. GITC irreversibly denatures proteins and RNases. This is followed by centrifugation.
Some sample sources present special challenges in RNA isolation or contain substances that cause problems in RNA analysis. These guides to RNA isolation have tips for a whole range of sample types, including guidance on the best kits for each.
Explain the basic mechanisms involved in DNA extraction.
Describe the steps involved in gDNA extraction from blood.
Explain the processes involved in quality and quantity check of extracted DNA using nanodrop technique.
Decribe the steps of quantity check of amplicon using flurometer.
Decribe the principle of dilution of amplicon.
Presented by,
Dr. Md. Mohiuddin Masum
Guided by,
Prof. Laila Anjuman Banu
methods of isolation and extraction of RNA by using different source such as plant tissues, bacterial culture, etc. Ribonucleic acid can be isolated from plant tissue for the purpose of:
– mRNA isolation
– In vitro translation
– Northern analysis
– cDNA library construction
Rigorous ribonuclease free environment is to be maintained
All glasswares, plasticwares and reagents made RNAse free (using 0.01% DEPC)
Next day, DEPC is inactivated by autoclaving for 30 min
Total RNA is isolated and separated from DNA and protein after extraction with a solution called as Trizol. Trizol is an acidic solution containing guanidinium thiocyanate (GITC), phenol and chloroform. GITC irreversibly denatures proteins and RNases. This is followed by centrifugation.
Some sample sources present special challenges in RNA isolation or contain substances that cause problems in RNA analysis. These guides to RNA isolation have tips for a whole range of sample types, including guidance on the best kits for each.
Explain the basic mechanisms involved in DNA extraction.
Describe the steps involved in gDNA extraction from blood.
Explain the processes involved in quality and quantity check of extracted DNA using nanodrop technique.
Decribe the steps of quantity check of amplicon using flurometer.
Decribe the principle of dilution of amplicon.
Presented by,
Dr. Md. Mohiuddin Masum
Guided by,
Prof. Laila Anjuman Banu
The 10 basic tips & tricks presented in this slide-deck are based on Frequently Asked Questions raised by scientists, and their answers as supplied by the Ambion technical support teams at Life Technologies.
The technique of molecular biology like DNA isolation, RNA isolation, PCR, Western blot, RFLP, etc was developed with development in science. This presentation includes the method of DNA and RNA isolation and their Quantification techniques.
Effective disruption of the biological matrix (cell, tissue, environmental or biological sample) to release the nucleic acids. Denaturation of structural proteins associated with the nucleic acids (nucleoproteins) Inactivation of nucleases that will degrade the isolated product (RNase and/or DNase).
Once the genomic DNA is bound to the silica membrane, the nucleic acid is washed with a salt/ethanol solution. These washes remove contaminating proteins, lipopolysaccharides and small RNAs to increase purity while keeping the DNA bound to the silica membrane column.
There are five basic steps of DNA extraction that are consistent across all the possible DNA purification chemistries:
disruption of the cellular structure to create a lysate,
separation of the soluble DNA from cell debris and other insoluble material,
binding the DNA of interest to a purification matrix,
washing proteins and other contaminants away from the matrix and
elution of the DNA.
Back to basics: Fundamental Concepts and Special Considerations in RNA IsolationQIAGEN
RNA integrity and quality are critical to obtain meaningful and reliable downstream data. This slidedeck details the challenges and considerations of handling RNA samples, RNA stabilization, the need for quality control analysis and common methods for RNA integrity and quality assessment.
As you know, Sample preparation is a relevant step to get the success in your research. For this reason, Canvax™ Extraction kits are designed for a reliable, easy and fast purification of high-quality and high-purity genomic DNA/RNA from a wide range of starting materials, like Blood, Buccal Swab, Buccal Saliva, Tissues, Cultured cells, Stool samples, Plant tissues, Soil samples, Bacteria, Yeast, Plasmid, PCR mixtures, Agarose gel slices or Serum.
Our breakthrough and proprietary technologies HigherPurity™, CleanEasy™ and WideUSE™ improves the most common Extraction methods conferring the obtained DNA and RNA a proven optimal performance for all downstream applications, such as PCR, qPCR, NGS, Cloning, STR Analysis or Gene Expression.
The 10 basic tips & tricks presented in this slide-deck are based on Frequently Asked Questions raised by scientists, and their answers as supplied by the Ambion technical support teams at Life Technologies.
The technique of molecular biology like DNA isolation, RNA isolation, PCR, Western blot, RFLP, etc was developed with development in science. This presentation includes the method of DNA and RNA isolation and their Quantification techniques.
Effective disruption of the biological matrix (cell, tissue, environmental or biological sample) to release the nucleic acids. Denaturation of structural proteins associated with the nucleic acids (nucleoproteins) Inactivation of nucleases that will degrade the isolated product (RNase and/or DNase).
Once the genomic DNA is bound to the silica membrane, the nucleic acid is washed with a salt/ethanol solution. These washes remove contaminating proteins, lipopolysaccharides and small RNAs to increase purity while keeping the DNA bound to the silica membrane column.
There are five basic steps of DNA extraction that are consistent across all the possible DNA purification chemistries:
disruption of the cellular structure to create a lysate,
separation of the soluble DNA from cell debris and other insoluble material,
binding the DNA of interest to a purification matrix,
washing proteins and other contaminants away from the matrix and
elution of the DNA.
Back to basics: Fundamental Concepts and Special Considerations in RNA IsolationQIAGEN
RNA integrity and quality are critical to obtain meaningful and reliable downstream data. This slidedeck details the challenges and considerations of handling RNA samples, RNA stabilization, the need for quality control analysis and common methods for RNA integrity and quality assessment.
As you know, Sample preparation is a relevant step to get the success in your research. For this reason, Canvax™ Extraction kits are designed for a reliable, easy and fast purification of high-quality and high-purity genomic DNA/RNA from a wide range of starting materials, like Blood, Buccal Swab, Buccal Saliva, Tissues, Cultured cells, Stool samples, Plant tissues, Soil samples, Bacteria, Yeast, Plasmid, PCR mixtures, Agarose gel slices or Serum.
Our breakthrough and proprietary technologies HigherPurity™, CleanEasy™ and WideUSE™ improves the most common Extraction methods conferring the obtained DNA and RNA a proven optimal performance for all downstream applications, such as PCR, qPCR, NGS, Cloning, STR Analysis or Gene Expression.
This is an internship report on molecular biology techniques, which was performed at PERD center under the guidance of Dr. Anshu Srivastava. This pdf contains all the basic information which is a preliminary requisite to know while approaching the molecular biology experimentally.
PCR (polymerase chain reaction) and Extraction of DNA from fungal plant patho...AjayDesouza V
PCR, Polymerase chain reaction, types of PCR, Template DNA, DNA polymerase, Primers, Nucleotides (DNTPs or deoxynucleotide triphosphates ), Denaturation, Annealing, Extension, Types of PCR, Multiplex PCR.
Long-range PCR.
Single-cell PCR.
Fast-cycling PCR.
Methylation-specific PCR (MSP)
Hot start PCR
High-fidelity PCR.
RAPD: Rapid amplified polymorphic DNA analysis.
Detection of fungal plant pathogen using PCR, Extraction of DNA from plant tissues,PCR amplification and detection of diagnostic amplicon
A biochemical technique used in Molecular Biology to amplify a specific fragment of target DNA.
PCR is used in medical and biological research, including cloning, genetic analysis, genetic fingerprinting, diagnostics, pathogen detection and genetic fingerprinting
An improved method for RNA extraction from woody legume species Acacia koa A....Premier Publishers
It is difficult to extract high-quality RNA of sufficient quantity from the stem tissues of tree legumes, such as Acacia koa and Leucaena leucocephala, because they contain high amounts of phenolic compounds and polysaccharides. The objective of this study was to develop an improved protocol that produces high-quality RNA from the stem tissues of these tree legumes. We developed a modified method that utilizes a lysis buffer containing a mixture of two commercially available reagents, RLT buffer from Qiagen RNeasy Kit and Fruit-mate™ from Takara. Comparison of the modified method with four other RNA extraction methods (Qiagen RNeasy, Fruit-mate™, TRIzol, and cetyltrimethylammonium bromide (CTAB)) showed that the modified method was far superior to the other methods. The extracted RNA produced reproducible DNA products in reverse transcription-polymerase chain reaction (RT-PCR). This improved protocol is rapid and easy, and it will facilitate genetic studies of A. koa, L. leucocephala, and other woody plants.
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
As consumer awareness of health and wellness rises, the nutraceutical market—which includes goods like functional meals, drinks, and dietary supplements that provide health advantages beyond basic nutrition—is growing significantly. As healthcare expenses rise, the population ages, and people want natural and preventative health solutions more and more, this industry is increasing quickly. Further driving market expansion are product formulation innovations and the use of cutting-edge technology for customized nutrition. With its worldwide reach, the nutraceutical industry is expected to keep growing and provide significant chances for research and investment in a number of categories, including vitamins, minerals, probiotics, and herbal supplements.
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
Observation of Io’s Resurfacing via Plume Deposition Using Ground-based Adapt...Sérgio Sacani
Since volcanic activity was first discovered on Io from Voyager images in 1979, changes
on Io’s surface have been monitored from both spacecraft and ground-based telescopes.
Here, we present the highest spatial resolution images of Io ever obtained from a groundbased telescope. These images, acquired by the SHARK-VIS instrument on the Large
Binocular Telescope, show evidence of a major resurfacing event on Io’s trailing hemisphere. When compared to the most recent spacecraft images, the SHARK-VIS images
show that a plume deposit from a powerful eruption at Pillan Patera has covered part
of the long-lived Pele plume deposit. Although this type of resurfacing event may be common on Io, few have been detected due to the rarity of spacecraft visits and the previously low spatial resolution available from Earth-based telescopes. The SHARK-VIS instrument ushers in a new era of high resolution imaging of Io’s surface using adaptive
optics at visible wavelengths.
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
Slide 1: Title Slide
Extrachromosomal Inheritance
Slide 2: Introduction to Extrachromosomal Inheritance
Definition: Extrachromosomal inheritance refers to the transmission of genetic material that is not found within the nucleus.
Key Components: Involves genes located in mitochondria, chloroplasts, and plasmids.
Slide 3: Mitochondrial Inheritance
Mitochondria: Organelles responsible for energy production.
Mitochondrial DNA (mtDNA): Circular DNA molecule found in mitochondria.
Inheritance Pattern: Maternally inherited, meaning it is passed from mothers to all their offspring.
Diseases: Examples include Leber’s hereditary optic neuropathy (LHON) and mitochondrial myopathy.
Slide 4: Chloroplast Inheritance
Chloroplasts: Organelles responsible for photosynthesis in plants.
Chloroplast DNA (cpDNA): Circular DNA molecule found in chloroplasts.
Inheritance Pattern: Often maternally inherited in most plants, but can vary in some species.
Examples: Variegation in plants, where leaf color patterns are determined by chloroplast DNA.
Slide 5: Plasmid Inheritance
Plasmids: Small, circular DNA molecules found in bacteria and some eukaryotes.
Features: Can carry antibiotic resistance genes and can be transferred between cells through processes like conjugation.
Significance: Important in biotechnology for gene cloning and genetic engineering.
Slide 6: Mechanisms of Extrachromosomal Inheritance
Non-Mendelian Patterns: Do not follow Mendel’s laws of inheritance.
Cytoplasmic Segregation: During cell division, organelles like mitochondria and chloroplasts are randomly distributed to daughter cells.
Heteroplasmy: Presence of more than one type of organellar genome within a cell, leading to variation in expression.
Slide 7: Examples of Extrachromosomal Inheritance
Four O’clock Plant (Mirabilis jalapa): Shows variegated leaves due to different cpDNA in leaf cells.
Petite Mutants in Yeast: Result from mutations in mitochondrial DNA affecting respiration.
Slide 8: Importance of Extrachromosomal Inheritance
Evolution: Provides insight into the evolution of eukaryotic cells.
Medicine: Understanding mitochondrial inheritance helps in diagnosing and treating mitochondrial diseases.
Agriculture: Chloroplast inheritance can be used in plant breeding and genetic modification.
Slide 9: Recent Research and Advances
Gene Editing: Techniques like CRISPR-Cas9 are being used to edit mitochondrial and chloroplast DNA.
Therapies: Development of mitochondrial replacement therapy (MRT) for preventing mitochondrial diseases.
Slide 10: Conclusion
Summary: Extrachromosomal inheritance involves the transmission of genetic material outside the nucleus and plays a crucial role in genetics, medicine, and biotechnology.
Future Directions: Continued research and technological advancements hold promise for new treatments and applications.
Slide 11: Questions and Discussion
Invite Audience: Open the floor for any questions or further discussion on the topic.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
Comparative structure of adrenal gland in vertebrates
molecular biology techniques -jaypee university of information technology- ravi ranjan
1. Under the supervision of – Prof. (Dr) R.S. Chauhan
Jaypee University of Information Technology
Presented by- Ravi Ranjan
B.Sc. Biotechnology (H)
Sem: V
Exam Roll No.: 12SBIT020909
Class Roll No.: 708
2. Experiments
performed:-
DNA isolation from Jatropha curcas plant sample
RNA isolation form Jatropha curcas plant sample
Determination of quality and quantity of isolated DNA and RNA
Polymerase chain reaction (PCR)
Cloning of Amplified fragment :-
Isolation of plasmid DNA
Ligation
Competent cell preparation
Transformation
Screening
Recombinant plasmid isolation
3. Jatropha curcas
Jatropha is poisonous, semi evergreen
shrub (6m tall).
They are also grown in deserts.
Seeds of jatropha are rich in oil, which
might be a new source of bio fuel.
DNA or RNA isolation was performed
using the leaf of the same plant.
In cloning also the genes this pant was
used ( DGAT, PAP, LPAT, GPAT) which
are involved in tri-acyl glycerol pathway
(Bio-Diesel production pathway).
4. DNA ISOLATION BY
C TAB METHOD
DNA is a higher molecular weight
molecule and can be isolated from
plant tissue by fracturing cell wall,
and cell membrane using a
detergent and it can be purified
from other contaminants using
P:C:I.
The isolation of plant nucleic acid
is a fundamental requirement for
most characterization, for
identification and isolation of
genes from genetic material.
DNA isolation from Jatropha
curcas plant sample
RNA isolation form Jatropha
curcas plant sample
Determination of quality
and quantity of isolated
DNA and RNA
Polymerase chain reaction
(PCR)
Cloning of Amplified
fragment
5. C TAB method of DNA isolation
Leaf sample is taken, grinded, and taken in micro-centrifuge tube,
pre-warmed C TAB is added, and RNase.
Incubated at 66˚C for 1 hour.
C:I added, mixed, centrifuged at 1000rpm for 10 minutes, aqueous
phase is transferred to another tube.
Isopropanol and sodium acetate is added and mixed well, kept at
20 ˚C for 1 hour or overnight.
Centrifuged at 10000rpm for 10 minutes, pellet is retained and
washed using ethanol.
6. RNA ISOLATION BY
C TAB METHOD
RNA can be isolated from plant
tissue by C TAB method.
The isolation of RNA is
preferred over DNA in genomic
laboratories or banks, since
cDNA synthesis and library
preparation is easy to maintain
because it doesn’t have interons.
DNA isolation from Jatropha
curcas plant sample
RNA isolation form Jatropha
curcas plant sample
Determination of quality
and quantity of isolated
DNA and RNA
Polymerase chain reaction
(PCR)
Cloning of Amplified
fragment
7. C TAB method of RNA isolation
Leaf sample is taken, grinded, and taken in micro-centrifuge tube, extraction
buffer is added, and RNase.
Incubated at 65˚C for 30 minutes.
C:I added, mixed, centrifuged at 12’000rpm for 10 minutes, aqueous phase is
transferred to another tube.
Upper phase was taken, C:I is added again centrifuged at 1000rpm.
Aqueous phase is taken, Isopropanol is added and centrifuged at 12’000 rpm .
Pellet is washed using ethanol (centrifuged at 12’000rpm) and air dried, and
retained in nuclease free water.
8. Determination of quality and quantity of
isolated
DNA and RNA
The quality and quantity of the genomic
DNA/RNA was checked using AGE. Good
quality of DNA/RNA shows intact band
without smearing.
The purity and concentration
of DNA/RNA was checked
using NANODROP
spectrophotometer at OD
260/280 taken against TE or
nuclease free water as blank.
The DNA or RNA sample showing the OD
260/280 value between 1.7-1.9 was
considered as pure sample and the
concentration of genomic DNA was
estimated.
DNA isolation from Jatropha
curcas plant sample
RNA isolation form Jatropha
curcas plant sample
Determination of quality
and quantity of isolated
DNA and RNA
Polymerase chain reaction
(PCR)
Cloning of Amplified
fragment
9. Casting of Agarose gel
The casting unit was setup.
0.8% Agarose DNA, and 1.2% RNA Agarose is dissolved in
100ml of 1x TAE.
Heated to dissolve and 4µl of EtBr is added before
cooling.
Poured in tray and comb was placed.
Left for solidification at room temp. for 30 minutes.
10.
11. DNA and RNA bands observed in UV Transilluminator
12. Polymerase chain reaction (PCR)
DNA isolation from Jatropha
curcas plant sample
RNA isolation form Jatropha
curcas plant sample
Determination of quality
and quantity of isolated
DNA and RNA
Polymerase chain reaction
(PCR)
Cloning of Amplified
fragment
A biochemical technology used to amplify a
single or a few copies of a piece of DNA across
several orders of magnitude, generating
thousands or millions of copies of a particular
DNA sequence.
The process mainly involves three basic stages:
Denaturation: at 94-96˚C, the ds DNA
denatures into single separated strands.
Annealing: at 50-65˚C, two primers
anneal the complementary seq. on either
side of DNA.
Extension: at 70-72˚C, the polymerase
binds to and extends the complementary
DNA stand from each primer.
14. Cloning
DNA isolation from Jatropha
curcas plant sample
RNA isolation form Jatropha
curcas plant sample
Determination of quality
and quantity of isolated
DNA and RNA
Polymerase chain reaction
(PCR)
Cloning of Amplified
fragment
CLONE: A collection of molecules or cells which
are all identical to an original molecule and cell.
CLONING VECTOR: It is a DNA molecule in
which foreign DNA can be inserted and which is
capable of replicating within host cell to
produce multiple clones of recombinant DNA.
CLONING STEPS:-
Plasmid isolation
Ligation
Competent cell preparation
Transformation
Screening (blue white screening)
Recombinant plasmid isolation
15.
16. Isolation of plasmid DNA
(from E. coli)
The cells are centrifuged and, resuspended pellet in different
buffers(STE buffer, GTE buffer)after each centrifuge.
Lysozyme solution is added, mixed.
Solution ii and iii is added (double volume and 1/3rd)and incubated at
room temperature and ice respectively, and again centrifuged.
Supernatant is taken in fresh tube, added alcohol and refrigerated
overnight.
Centrifuged, washed with ethanol and dried.
Dissolved in TE buffer so that it can be used further.
17. Ligation
Reagents Volume used
5X ligation buffer 2µl
PGEM-T vector 1µl
T4 ligase 1µl
Sterile water Add to final volume of 10µl
Fresh PCR product 2-3µl
Final volume 10µl
The reaction was mixed gently and incubated at 4˚C for overnight.
18. Competent cell preparation
Single colony is picked up and grown overnight.
Pre-warmed algae media is added and stored at 37˚C, cooled
on ice and transferred to sterile centrifuge tube.
Centrifuge at 4000g for 5 min at 4 ˚C, supernatant is discarded
n tube is kept on ice.
Resuspended in 30 ml solution i containing RbCl2, KCH2COOH,
CaCl2 and glycerol for 90 minutes, and centrifuged it again and
pellet resuspended in 4 ml solution containing MOPS, RbCl2
e.tc.
19. Transformation
competent cells and DNA is added in eppendroff tubes, stored in ice for 3o
minutes, and then transferred to pre-heated water bath at 42˚C for 90
seconds.
Tubes are immediately transferred to ice box, and kept for 2 minutes.
incubated in water bath at 37˚C for 45 minutes, and is kept in shaker
incubator for 15 minutes.
Transformed cells are transferred on Petri plates containing algae agar
medium, and spread, incubated at 37˚C for 18 hours.
21. Recombinant plasmid isolation
white colonies are picked and inoculated in 3 ml LB broth and incubated
at 37 ˚C for overnight in shaker.
Cultures is centrifuged for 1 minute at 14000 rpm, and resuspended in
Tris and EDTA containing solution.
solution with NaOH, SDS, potassium acetate is added and mixed gently, a
white precipitate is observed.
Tube is centrifuged for 10 minutes at 14000rpm and supernatant is taken
in fresh tube.
Ethanol washing is done, air dried and suspended in sterile nuclease free
water or TE buffer and stored.