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TA cloning: A simple subcloning strategy
Short Description: Ligate a DNA fragment into a vector using TA cloning, make competent cells, transform cells, screen for recombinants using blue white screening and clone confirmation by restriction analysis.
Type: Hands-on
Duration: Two days
Fees: INR 5192 (INR 4400 + 18% GST)
Category (for payment): MBY Workshop Tier 4
Aimed at: College students (B.Sc. and M.Sc.)
Application Process: Click here for the details
Registration form (This link will be different for each workshop)
Application process (in brief)
Application process (in brief)
Check the above table to figure out whether suitable dates are available or not.
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Course Content
Course Content
1. Basic laboratory practices
1. Basic laboratory practices
Basic safety, handling of micropipettes and safe disposal of laboratory wastes. To know more about how to handle micropipettes, watch this video or practice using this simulation.
2. Polymerase Chain Reaction (PCR)
2. Polymerase Chain Reaction (PCR)
Once the entire DNA has been isolated, often a specific region of interest needs to be isolated form it. The simplest way to do this is through the technique of Polymerase Chain Reaction (PCR) which amplifies the number of copies of the DNA of interest. To know more about PCR, you can either read this article.
3. Gel Electrophoresis
3. Gel Electrophoresis
Once the PCR step is done, one needs to confirm whether the amplification has happened properly or not. Perform the technique of gel electrophoresis for confirmation. To know more about gel electrophoresis, you can read this article.
4. Ligation
4. Ligation
DNA fragments with A overhangs are joined with vector plasmids having compatible T overhangs with the help of the enzyme DNA ligase. To know more, read this article on TA cloning.
5. Competent cell preparation, transformation and plating
5. Competent cell preparation, transformation and plating
Bacterial cells are chemically treated to prepare them for taking up the naked DNA. Transformation is performed by heat shock and cells are plated onto antibiotic containing agar plates. The total number of colonies are counted to calculate the efficiency of the chemically competent cells to take up the DNA. Read the article on competent cells and transformation.
6. Blue-white screening, clone analysis and confirmation
6. Blue-white screening, clone analysis and confirmation
Colonies that grow after transformation are screened via blue-white technique and confirmed for the presence of DNA of interest through plasmid DNA isolation and restriction digestion. To understand the principle of blue-white screening, watch this video.
7. Isolation of plasmid DNA by alkaline lysis
7. Isolation of plasmid DNA by alkaline lysis
Plasmid DNA extraction is a critical step in confirming whether the cloning was successful in the screened white colonies and there are no false positives. It is essential for procedures like gene cloning. Read about alkaline lysis here.
8. Diagnostic Restriction Digestion
8. Diagnostic Restriction Digestion
This technique makes use of restriction endonucleases to cut the plasmid DNA into smaller fragments which can then be run on an agarose gel. We check the size of the fragments to confirm whether the gene cloning is successful or not. To know more about restriction enzymes, read this article.
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