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.

110 µl           5 µl   10 x Taq buffer(see below)  11 µl         0.5 µl   20 mM dNTP's (0.2 mM) 11 µl         0.5 µl   Taq Polymerase (2.5 units)638 µl   22 X   29 µl   water                 5 µl   10 µM upstream primer: A,C,or kanC (1 µM)                 5 µl   10 µM downstream primer:B,kanB,or D(1 µM)     5 µl   Zymolyase treated cells                50 µl total volume

*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:

                     3 min, 94 °C (initial denaturation)              --->   15 sec, 94 °C   35 cycles: --->   15 sec, 57 °C              --->   60 sec, 72 °C                     3 min, 72 °C (final elongation)

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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