Griffitts:PCR

PCR with Taq Polymerase

Procedure

Prepare your template sample
- For amplifying rhizobium 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 buffer, dNTPs and primers
- Keep polymerase on ice throughout the procedure
In standard PCR tube combine components according to the recipe below
- Set the reaction up on ice
- When running multiple samples, a Master Mix, which includes everything except the except the template and the primers (add 43.4 μL Master Mix to each tube)
- If performing multiple PCRs with the same primers then just use 100 μM stock, calculate for 0.3 μL per reaction, and correct the volume of water
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

37 μl H₂O
5 μl 10X Taq buffer
1 μl dNTPs
0.4 μl Taq polymerase
1 μl template
3 μl forward primer
3 μl reverse primer