Griffitts:PCR
From OpenWetWare
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 | Per reaction |
---|---|
dH2O | 17.8 μL |
10X Taq buffer | 2.5 μL |
dNTPs | 0.5 μL |
Taq polymerase | 0.2 μL |
DNA template | 1 μL |
forward primer | 1.5 μL diluted 1:10 |
reverse primer | 1.5 μL diluted 1:10 |
40 μL reactions
This is the standard recipe. Use it in most cases for cloning.
Ingredient | Per reaction |
---|---|
dH2O | 29.1 μL |
10X Taq buffer | 4 μL |
dNTPs | 0.8 μL |
Taq polymerase | 0.3 μL |
DNA template | 1 μL |
forward primer | 2.4 μL diluted 1:10 |
reverse primer | 2.4 μL diluted 1:10 |
40 μL reaction with Pfx polymerase
Use this for high-fidelity cloning.
Ingredient | Per reaction |
---|---|
dH2O | 27.4 μL |
10X Pfx buffer | 4.0 μL |
dNTPs | 1.2 μL |
Pfx polymerase | 1.0 μL |
DNA template | 1.6 μL |
forward primer | 2.4 μL diluted 1:10 |
reverse primer | 2.4 μL diluted 1:10 |
Master Mix Calculations
Ingredient | 25 μL reaction
with Taq polymerase |
40 μL reaction
with Taq polymerase |
40 μL reaction
with Pfx polymerase |
---|---|---|---|
dH2O | 20.5 μL | 33.4 μL | 32.3 |
10X Taq buffer | 2.5 μL | 4.0 μL | 4.0 μL |
dNTPs | 0.5 μL | 0.8 μL | 1.2 μL |
polymerase | 0.2 μL | 0.3 μL | 1.0 μL |
forward primer | 0.15 μL undiluted | 0.24 μL undiluted | 0.24 μL undiluted |
reverse primer | 0.15 μL undiluted | 0.24 μL undiluted | 0.24 μL undiluted |
Total volume (without DNA) | 24 μL | 39 μL | 38.4 μL |
Primer Dilution
- 27 μL dH2O
- 3 μL primer
Repeat for both primers