Difference between revisions of "PCR Amplification of DNA"

Revision as of 16:49, 8 June 2020

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

• Buffer, 5 uL of 10X buffer (Dave's fridge)
• Primers, 10uL of 1uM stock solution in water (both primers combined)
• dNTPs, 5uL of 2 mM ("molecular biology stuff" box in freezer)
• Sterile water, 28 uL
• Template 1 uL
• Polymerase 1 uL (turbo pfu found in "enzymes" box in freezer)

• 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)