Conventional PCR amplification requires multiple steps of bacterial culture and plasmid preparation before gene amplification, which is cumbersome and time-consuming, and the amount of DNA lost during repeated operations is also large, resulting in a low yield. In 1989, Gussow Clackson established the colony PCR method, which is different from the ordinary DNA PCR in that it directly uses a single bacterium as a template. It is a method that can quickly identify whether the colony is positive for the target plasmid or not, and it is simple, fast and commonly used in transformation identification.
Principle
The basic principle of colony PCR is to use a single colony as a template, use a sterile toothpick to pick a single colony into TE buffer, boil for 10 min, vortex and oscillate, then centrifuge briefly, and then use 1~2 countries' lysate as a DNA template, which can eliminate the step of extracting template DNA, and then amplify the target genes by specific primers or universal primers, which can greatly save the time and cost.
Appliance
The common applications of [colony PCR] are as follows: 1. Pick recombinant clones Conventional alkaline lysis and boiling methods (Sambrook, 1989) require overnight incubation of the organisms, lysis of the organisms, and ethanol precipitation. Generally, only a small portion of the picked clones are recombinants, so the extraction of plasmids consumes a lot of time, while the process of picking recombinant clones by colony PCR avoids the tedious process of plasmid extraction. Chen Shuxia et al. used single colony PCR to directly screen recombinant clones containing green fluorescent protein (GFP) and exogenous genes of heat-resistant enterotoxin B subunit and heat-resistant enterotoxin fusion gene (LTEST), and the positive clones were able to amplify the target bands, which was in agreement with the results of plasmid PCR amplification. The results were consistent with those of plasmid PCR amplification, which proved that single colony PCR was very effective in identifying positive recombinant clones.2. Genome sequencingTraditional PCR amplification before sequencing is based on the DNA extracted by alkaline cleavage as template, which has a high success rate, but is more cumbersome. By using colony PCR method, the sample can skip the step of DNA extraction, and directly use the DNA exposed by pyrolysis of the bacterium as the template for PCR amplification and fluorescent labeling, which can reduce the total cost of the steps from library construction to sequencing by 1/3. Tang Yesheng et al. successfully determined the full-length sequence of BAC DNA of L3173 from Oryg sativa indica (Oryg sativa indica) Guanglu Dwarf No. 4 by using the method. The full-length BAC DNA sequence of L3173 of glazed rice (Oryg sativa indica) Guanglu Dwarf 4 was successfully determined by this method.
Operation method
Colony PCR experiment
Principle
The basic principle of colony PCR is to use a single colony as a template, pick a single colony with a sterile toothpick and put it into TE buffer, boil for 10 min, vortex and oscillate, then centrifuge briefly, and then use the lysate of 1~2 countries as a DNA template, eliminating the step of extracting the template DNA, and then amplifying the target genes by specific primers or universal primers, which can save the time and cost greatly.
Materials and Instruments
Equipment: Move 1. Pick a single colony with an autoclaved toothpick and draw a line on the plate containing resistance for seed preservation, then place the toothpick in 20 μL Triton × 100 and stir it to suspend the bacteria on the toothpick, and mark the corresponding bacteria and the drawn line area on the plate. 2. Boil the EP tube containing 20 μL of Triton × 100 for 10 min, cool it down and centrifuge it at 12000 r/min for 1 min to remove the cell debris. 3. Add the reaction system (25 μL) according to the expected plasmid content: 4、PCR amplification conditions: 95 ℃ for 5 min; 94 ℃ for 30 s, 55 ℃ for 30 s, 72 ℃ for 60 s, 30 cycles; 72 ℃ for 5 min; 4 ℃ storage. 5、Electrophoresis, observe the results, and preserve the colonies on the main plate corresponding to the positive results or sequencing. Caveat 1, colony PCR, add the bacterial template volume should not be too large, otherwise it will affect the PCR system, resulting in amplification failure, generally take 1~2 μL to make 25 μL of PCR system. If there are too many templates, the primers will not be enough, obviously not amplified, this phenomenon in the plasmid template PCR inside a lot of plasmids, do not forget to dilute the proposed plasmid. 2， reduce the amplification cycle, 25 ~ 30 cycles for colony PCR is more appropriate, amplification cycle is too much, will produce false positives.Control of false positives. If the carrier primers to amplify the general can reduce false positives, or more pairs of different primers amplification, in order to increase the screening results of true positives, PCR results are best to do the enzyme identification. 4. Control of PCR conditions. Hot start can eliminate primer dimer, annealing temperature can be slightly lower. For more product details, please visit Aladdin Scientific website.
Single colonies cultured on solid plate medium, autoclaved toothpicks, resistant plates, EP tubes, centrifuge, etc.
Reagents:
10 × PCR buffer (100 mmol/L Tris-HCl, pH 8.3, 500 mmol/L KC1; 0. 1% gelatin; 15 mmol/L MgCl
2
), primers for target genes, Triton × 100.