Difference between revisions of "PCR Amplification of DNA"
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| + | ==SOP== | ||
| + | *[[SOP - Electrophoresis]] | ||
| + | |||
==Materials== | ==Materials== | ||
| − | *Primers – order from IDT prediluted to 100 mM in TE. Make working solution of 1 uM (100x) in water with both sense and antisense primers combined | + | *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== | ==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. Normally use touchdown PCR ('''DAVETD''') as follows: | + | *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) | *Purify PCR product if necessary using Qiagen kit (Add 5x PB) | ||
Latest revision as of 16:57, 8 June 2020
Contents
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)
