The following colony PCR protocol has been designed to be performed in individual reaction tubes. We usually test three colonies from each transformation along with a wild-type control. A series of five different PCR tests are performed on each colony to confirm both of the novel recombination junctions .
1. Clonal purification
- Pick three colonies from a transformation plate and streak each one out to single colonies on separate G418 containing plates (200mg/l). - Incubate at 30°C for two to three days. *This step reduces background by eliminating aborted transformants.
2. Zymolyase treatment
- Transfer a small portion of a well-isolated colony (~1 mm) into 50 µl solution containing 60 U/ml of Zymolyase (This solution is generated by resuspending 6 mg of 100T Zymolyase in 10 mls of water. Ordering information: Seikagaku Corp., #120493-1).
- Sterile yellow tips work great for this task. - Repeat this step for each of the 3 isolates and the wild-type control. - Incubate the Zymolyase solutions at 37 ° C for 30 minutes followed by 10 minutes at 95 ° C. *It is not necessary to remove the Zymolyase solution by pelleting the cells before the PCR. *The Zymolyase treated cells can be stored at 4 ° C for several days before being used as template for the colony PCR. However, fresher is probably better.
*The amount of cells that gets picked into the Zymolyase does not seem to be too critical - similar PCR results were obtained when 1 µl, 5 µl or 10 µl of the Zymo treated cells were used as template.
3. PCR reactions
- The lyophilized confirmation primers (~5-10 nmoles of each oligonuleotide) should be resuspended in 750 µl of TE (final concentration of ~10 µM). We use 5 µl of each primer in 50 µl PCR reactions (~ 1 µM final primer concentration). - Label 20 thin-wall PCR tubes (5 for each isolate) and add the following primer pairs: A-B, A-kanB, C-D, kanC-D, and A-D. Tubes 1-5 are for isolate #1, 6-10 are for isolate #2, ..., and tubes 16-20 are for the wild-type control. Each of the 20 tubes should contain 10 µl of the primer mix (5 µl of each primer). - Add the 5 µl of the Zymo treated cells to each of the 20 PCR reactions. For example, add 5 µl of the Zymo solution from isolate #1 to PCR tubes 1-5, 5 µl of the Zymo solution from isolate #2 to PCR tubes 6-10 and so on. - Make a PCR master mix by combining: 638 µl water, 110 µl 10 x Taq Buffer, 11 µl NTP's, and 11 µl Taq Polymerase. - Add 35 µl of the PCR mix to each of the 20 PCR tubes that already contain the 15 µl of primers and template.
110µl5µl10xTaqbuffer(seebelow)11µl0.5µl20mMdNTP's(0.2mM)11µl0.5µlTaqPolymerase(2.5units)638µl22X29µlwater5µl10µMupstreamprimer:A,C,orkanC(1µM)5µl10µMdownstreamprimer:B,kanB,orD(1µM)5µlZymolyasetreatedcells50µltotalvolume
*10 x Taq buffer contains: 100 mM Tris-HCl (pH 8.4), 500 mM KCl, 15 mM MgCl2. *The final concentrations are shown in parentheses. *The Taq Polymerase should be added last and PCR mixture should be kept on ice until the PCR is started. Alternatively, a "hot start" can be performed by adding the Taq polymerase to the individual tubes after the PCR mixtures have been heated to 94°C. Hot start improves the PCR results but this step is labor intensive and we have not found it to be necessary.
4. PCR conditions:
3min,94°C(initialdenaturation)--->15sec,94°C35cycles:--->15sec,57°C--->60sec,72°C3min,72°C(finalelongation)
5. Agarose gel electrophoresis
- Add 10 µl 6x loading buffer (30% glycerol, 50mM EDTA, 0.25% bromophenol blue) to the 50 µl PCR reaction. - Load 10 µl on a 1.5% agarose gel.
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