The colony PCR analysis of cloned recombinants assay can be applied to (1) analyze whether colonies produced from transformed cells contain recombinant insert fragments, and (2) identify the presence and orientation of the insert.
Operation method
Colony PCR analysis of cloned recombinants experiments
Principle
Colony PCR (PCR) can save time and effort by directly using the DNA exposed by pyrolysis of the bacterium as a template for PCR amplification without the need to extract genomic DNA and identify it by enzymatic digestion. Using the universal primers on the vector, recombinants are screened or analyzed by DNA sequencing. The size of the final PCR product is the size of the insert fragment between the universal primers of the vector.
Materials and Instruments
Colonies Move I. Materials Caveat 1. Designing primers is critical. Generally speaking, if it is a targeted clone, use the universal primer on the vector; for example, pET series can use T7 universal primer. If it is a non-directional cloning (such as single enzyme digestion or flat end joining), one primer is used with the vector and one primer is used on the target gene, so that it can be more convenient to identify, and the probability of error is very low. the selection of PCR conditions is close to the best, and at the same time, not too many bacteria should be picked, or there will be non-specific amplification. 2. The concentration of the primers used should not be too high, the concentration will lead to non-specific amplification, and the number of cycles of the reaction should not be too much, usually not more than 25. At the same time, because of the GC content of the amplified fragments, some GC content is very low, and some are very high, resulting in colony PCR is not easy to amplify the target band, it is recommended that when setting up the PCR program to the upper limit of the temperature of the high GC, each cycle down 0.2 degrees or so. Common Problems Toothpicks can carry away liquids by capillary action and therefore change the concentration of the reaction mixture. For more product details, please visit Aladdin Scientific website.
Ethidium bromide Taq Extension PCR Additives Tween20 10% solution dNTP solution PCR buffer Heat stabilized DNA polymerase Primers
Sterile tips Agarose gel electrophoresis equipment and reagents
1. Buffers, solutions and reagents
Ethidium bromide
Taq Extension PCR Additive (Stratagene)
Tween 20, 10% solution
2. Enzyme and enzyme buffer
dNTP solution (contains all 4 dNTPs, 25 mmol/L each)
PCR buffer, 10X, provided by the company when the user buys the heat-stabilized polymerase, or PCR optimization buffer, e.g., Opti-Prime PCR Optimization Kit (Stratagene), and PCR Optimizer (Invitrogen).
Heat stabilized DNA polymerase (5-10U), e.g., Taq DNA polymerase, cloned Pfu DNA polymerase (Stratagene)
3. Nucleic acids and oligonucleotides
Optional: insert-specific primers (used to orient the insert in bidirectional cloning)
A set of primers to screen for recombinants
4. Specialized equipment
Sterile tips for scribing and splicing of bacteria
The reason for using a lance tip instead of a toothpick is that a toothpick can carry away liquid by capillary action and therefore change the concentration of the reaction mixture.
5. Additional items
Agarose gel electrophoresis equipment and reagents including ethidium bromide
II. METHODS
1. Depending on the number of reactions or tubes of PCR reactions to be done, prepare a cocktail on ice using a centrifuge tube like
Prepare the PCR "cocktail" style mixture in a centrifuge tube on ice by adding the ingredients in the following order.
H2O to make up the final volume to 25ul
Taq buffer, 10X 2.5ul
Tween20 (10% solution by volume) 1.0ul
dNTP mixture (25 mmol/L of each dNTP) 0.2ul
Recombinant screening primer set (100ng/ul) 1ul
Taq Extension PCR Additive (5U/ul) 0.25ul
Taq DNA Polymerase (5U/ul) 0.25ul
2. On ice, dispense 25ul of the "cocktail" PCR mixture into each PCR tube.
3. For the control reaction, add 1ul of non-recombinant DNA.
Optional: For reactions used to determine insertion direction, add a third insert-specific primer to the appropriate reaction tube.
4. for the recombination screen, prick the transformed colonies with a sterile lance tip and stir the colony material in the appropriate reaction tube. After inoculating each reaction mixture, quickly remove the lance tip and lightly coat an antibiotic-containing plate to retain the strain for subsequent analysis. For PCR-mediated recombination screening, also see the troubleshooting below.
5. Gently mix each reaction system.
6. Perform PCR with the recommended cycling parameters.
7. Analyze PCR products by standard agarose gel electrophoresis. 10-15 g/L agarose gels are recommended to optimize the resolution of 500-6000 bp PCR products. Agarose gels of 10-15 g/L are recommended to optimize the resolution of PCR products of 500-6000 bp. Typically, 1 to 5 ul of PCR product is run through a bromoform. Typically, 1 to 5 ul of PCR product is analyzed by staining with ethidium bromide. Images can be acquired by conventional instantaneous imaging techniques or by computerized imaging software.