PCR Amplification of DNA

SOP

• SOP - Electrophoresis

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. 1 min at 94
2. 30s at 65
3. 2 min/kb at 72
4. 30s at 94
5. 30s at 63 then -0.5/cycle
6. 2 min/kb at 72
7. Repeat steps 4-6 28 times
8. 30s at 94
9. 30s at 45
10. 11 min at 72
11. Hold at 4 until ready

• Purify PCR product if necessary using Qiagen kit (Add 5x PB)