PCR Amplification of DNA
SOP
Materials
- Primers – order from IDT prediluted to 100 mM in TE. Make working solution of 0.4uM-1 uM (100x) in water with both sense and antisense primers combined (300uL water, 3uL of each primer)
- DreamTaq Green PCR Master mix - contains dNTPs, polymerase, salts, and buffer with loading dye https://www.thermofisher.com/order/catalog/product/K1081?ICID=search-product#/K1081?ICID=search-product
- RNAse-Free water - comes in DreamTaq kit
Protocol
- Use the following volumes per reaction
- 12.5 uL DreamTaq
- 7.5 uL RNAse-Free water
- 5.0 uL Working stock Primer (of 0.4-1uM stock solution in water (both primers combined))
PCR Program
- For animals: use programs denoted in Genotyping Program
- Run PCR Program (approx 3.5 to 4 hours). Normally use touchdown PCR (DAVETD) as follows:
- 1 min at 94
- 30s at 65
- 2 min/kb at 72
- 30s at 94
- 30s at 63 then -0.5/cycle
- 2 min/kb at 72
- Repeat steps 4-6 28 times
- 30s at 94
- 30s at 45
- 11 min at 72
- Hold at 4 until ready
- Purify PCR product if necessary using Qiagen kit (Add 5x PB)