Difference between revisions of "PCR Amplification of DNA (KOD Polymerase)"
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*dNTPs – dilute to 2 mM each in water, make single use aliquots (50 uL aliquots... 10uL of each dNTP, 460uL water) | *dNTPs – dilute to 2 mM each in water, make single use aliquots (50 uL aliquots... 10uL of each dNTP, 460uL water) | ||
*Template – generally 1uL or less of a plasmid miniprep | *Template – generally 1uL or less of a plasmid miniprep | ||
| − | *Polymerase | + | *KOD Polymerase |
==Protocol== | ==Protocol== | ||
| Line 16: | Line 16: | ||
*Run PCR Program (approx 3.5 to 4 hours). Normally use touchdown PCR ('''TD-KOD''') as follows: | *Run PCR Program (approx 3.5 to 4 hours). Normally use touchdown PCR ('''TD-KOD''') as follows: | ||
| − | :# | + | :#15s at 94 |
| − | :# | + | :#5s at 65 |
| − | :# | + | :#30s at 72 |
| − | :# | + | :#15s at 94 |
| − | :# | + | :#5s at 63 then -0.5/cycle |
| − | :# | + | :#30s at 72 |
| − | :#Repeat steps 4-6 | + | :#Repeat steps 4-6 30 times |
:#30s at 94 | :#30s at 94 | ||
:#30s at 45 | :#30s at 45 | ||
Latest revision as of 21:24, 3 November 2009
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 (300uL water, 3uL of each primer)
- dNTPs – dilute to 2 mM each in water, make single use aliquots (50 uL aliquots... 10uL of each dNTP, 460uL water)
- Template – generally 1uL or less of a plasmid miniprep
- KOD Polymerase
Protocol
- Use the following volumes per reaction
- KOD Buffer #1, 5 uL of 10X buffer (Dave's Molecular Biology Stuff Box)
- Primers, 10uL of 1uM stock solution in water (both primers combined)
- dNTPs, 5uL of 2 mM (Dave's Molecular Biology Stuff Box)
- MgCl, 3uL of 3 mM (Dave's Molecular Biology Stuff Box)
- Sterile water, 28 uL
- Template 1 uL of a miniprep
- Polymerase 0.4 uL (KOD Polymerase found in "Enzymes" box in freezer)
- Run PCR Program (approx 3.5 to 4 hours). Normally use touchdown PCR (TD-KOD) as follows:
- 15s at 94
- 5s at 65
- 30s at 72
- 15s at 94
- 5s at 63 then -0.5/cycle
- 30s at 72
- Repeat steps 4-6 30 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)
