Lidstrom:Colony PCR

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(General:)
(General:)
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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.  
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*The primer concentration varies in different people's recipes.
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*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.   
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*Do rxns in 10-25 uL.  Too small of a volume can result in overloading without noticing.
 
*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.     
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*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]
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*[[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]
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==Parameters:==
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*T_anneal:
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*t_anneal: use 30s/kb and round up.
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*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 18:38, 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!
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