Griffitts:PCR
PCR with Taq Polymerase
Procedure
- Prepare your template sample
- For amplifying Sinorhizobium sequences, boil a dense suspension of cells in 35 μL PCR lysis buffer for ~2 min and vortex
- For amplifying E. coli, adding cells directly to the reaction is sufficient
- Thaw the Taq buffer, dNTPs, and primers
- Keep Taq polymerase on ice throughout the procedure
- Combine components according to one of the recipes below
- Set the reaction up on ice
- Select the appropriate cycling program and verify that all of the parameters are correct
- Allow 1 minute of extension time per 1 KB DNA being amplified
- Select "Run program" and select "YES" when asked about heated lid
- Wait until block has reached the beginning temperature. Place PCR tube into the machine
- Lower the lid until it latches and slightly tighten the heated lid onto the tubes
NOTE: When the PCR machine has finished cycling, your samples may be stored at -20°C. You may now proceed to gel electrophoresis and/or PCR clean-up.
Reaction recipes
25 μL reactions
Use this for diagnostic purposes.
| Ingredient | 2 reactions* | 3 or 4 reactions† | 5 or more reactions‡ |
|---|---|---|---|
| dH2O | 17.8 μL | 19.3 μL | 20.5 μL |
| 10X Taq buffer | 2.5 μL | 2.5 μL | 2.5 μL |
| dNTPs | 0.5 μL | 0.5 μL | 0.5 μL |
| Taq polymerase | 0.2 μL | 0.2 μL | 0.2 μL |
| DNA template | 1 μL | 1 μL | 1 μL |
| forward primer | 1.5 μL diluted 1:10 | 1.5 μL Primer Master Mix | 0.15 μL undiluted |
| reverse primer | 1.5 μL diluted 1:10 | (see above) | 0.15 μL undiluted |
* Make in separate tubes.
† Make a Reaction Master Mix by multiplying the amounts for each ingredient by one more than the number of reactions you are performing. Do not add the Primer Master Mix to the Reaction Master Mix. Add 22.5 μL of the Master Mix to each tube before adding the Primer Master Mix or the DNA template.
‡ Make a Reaction Master Mix by multiplying the amounts for each ingredient by two more than the number of reactions you are performing. Include the primers in the Reaction Master Mix. Add 24 μL of the Master Mix to each tube before adding the DNA template.
40 μL reactions
This is the standard recipe. Use it in most cases for cloning.
| Ingredient | 2 reactions* | 3 or 4 reactions† | 5 or more reactions‡ |
|---|---|---|---|
| dH2O | 29.1 μL | 31.5 μL | 33.42 μL |
| 10X Taq buffer | 4 μL | 4 μL | 4 μL |
| dNTPs | 0.8 μL | 0.8 μL | 0.8 μL |
| Taq polymerase | 0.3 μL | 0.3 μL | 0.3 μL |
| DNA template | 1 μL | 1 μL | 1 μL |
| forward primer | 2.4 μL diluted 1:10 | 2.4 μL Primer Master Mix | 0.24 μL undiluted |
| reverse primer | 2.4 μL diluted 1:10 | (see above) | 0.24 μL undiluted |
* Make in separate tubes.
† Make a Reaction Master Mix by multiplying the amounts for each ingredient by one more than the number of reactions you are performing. Do not add the Primer Master Mix to the Reaction Master Mix. Add 36.6 μL of the Master Mix to each tube before adding the Primer Master Mix or the DNA template.
‡ Make a Reaction Master Mix by multiplying the amounts for each ingredient by two more than the number of reactions you are performing. Include the primers in the Reaction Master Mix. Add 39 μL of the Master Mix to each tube before adding the DNA template.
50 μL reactions
| Ingredient | 2 reactions* | 3 or 4 reactions† | 5 or more reactions‡ |
|---|---|---|---|
| dH2O | 36.6 μL | 39.6 μL | 42 μL |
| 10X Taq buffer | 5 μL | 5 μL | 5 μL |
| dNTPs | 1 μL | 1 μL | 1 μL |
| Taq polymerase | 0.4 μL | 0.4 μL | 0.4 μL |
| DNA template | 1 μL | 1 μL | 1 μL |
| forward primer | 3 μL diluted 1:10 | 3 μL Primer Master Mix | 0.3 μL undiluted |
| reverse primer | 3 μL diluted 1:10 | (see above) | 0.3 μL undiluted |
* Make in separate tubes.
† Make a Reaction Master Mix by multiplying the amounts for each ingredient by one more than the number of reactions you are performing. Do not add the Primer Master Mix to the Reaction Master Mix. Add 46 μL of the Master Mix to each tube before adding the Primer Master Mix or the DNA template.
‡ Make a Reaction Master Mix by multiplying the amounts for each ingredient by two more than the number of reactions you are performing. Include the primers in the Reaction Master Mix. Add 49 μL of the Master Mix to each tube before adding the DNA template.
Primer Dilution
Standard Primers
- 27 μL dH2O
- 3 μL primer
Repeat for both primers
Primer Master Mix
- 24 μL dH2O
- 3 μL forward primer
- 3 μL reverse primer
