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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.


			Single tube confirmation PCR protocol 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. - see example last updated January 2002 amchu@cmgm.stanford.edu
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Single tube confirmation PCR protocol

1. Clonal purification

2. Zymolyase treatment

3. PCR reactions

4. PCR conditions:

5. Agarose gel electrophoresis