Lidstrom:Colony PCR: Difference between revisions
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*The thermocycling denaturation & extension temperature should be that recommended by your particular PCR kit. I have seen 68oC & 72oC. | *The thermocycling denaturation & extension temperature should be that recommended by your particular PCR kit. I have seen 68oC & 72oC. | ||
*The annealing temperature is up to you. For VF2 & VR 54-56oC works well. | *The annealing temperature is up to you. For VF2 & VR 54-56oC works well. | ||
*The primer concentration varies in different people's recipes. | *The primer concentration varies in different people's recipes. [[Janet B. Matsen|Janet]] hasn't tested variations. | ||
*One fatal flaw: overloading the PCR. Just a tiny piece of a colony should be used. | *One fatal flaw: overloading the PCR. Just a tiny piece of a colony should be used. | ||
*If they aren't working, you can put them in tubes, put them at 95oC for a while, then centrifuge they lysed cells so all the debris falls out. Run PCR from on the aqueous portion. | *If they aren't working, you can put them in tubes, put them at 95oC for a while, then centrifuge they lysed cells so all the debris falls out. Run PCR from on the aqueous portion. | ||
*Janet is using NEB's [http://www.neb.com/nebecomm/products/productM0486.asp OneTaq] quick-load (not hot start) & their recommended [http://www.neb.com/nebecomm/appNotes/appnoteM0486.pdf|protocol protocol] | *[[Janet B. Matsen|Janet]] is using NEB's [http://www.neb.com/nebecomm/products/productM0486.asp OneTaq] quick-load (not hot start) & their recommended [http://www.neb.com/nebecomm/appNotes/appnoteM0486.pdf|protocol protocol] | ||
==Parameters:== | |||
*T_anneal: | |||
*t_anneal: use 30s/kb and round up. | |||
*rxn volume: 10-25 uL. Too small of a volume can result in overloading without noticing. | |||
*Stay tuned for an image of what "overloading" can look like! | *Stay tuned for an image of what "overloading" can look like! | ||
Revision as of 16:38, 2 February 2012
Back to Protocols
General:
- Use Taq. It is cheap. Do not use Phusion. It is ~$1/50uL rxn. Taq is less accurate & less fast but that's fine.
- The thermocycling denaturation & extension temperature should be that recommended by your particular PCR kit. I have seen 68oC & 72oC.
- The annealing temperature is up to you. For VF2 & VR 54-56oC works well.
- The primer concentration varies in different people's recipes. Janet hasn't tested variations.
- One fatal flaw: overloading the PCR. Just a tiny piece of a colony should be used.
- If they aren't working, you can put them in tubes, put them at 95oC for a while, then centrifuge they lysed cells so all the debris falls out. Run PCR from on the aqueous portion.
Parameters:
- T_anneal:
- t_anneal: use 30s/kb and round up.
- rxn volume: 10-25 uL. Too small of a volume can result in overloading without noticing.
- Stay tuned for an image of what "overloading" can look like!
