Difference: ProtocolsStandardPCR (17 vs. 18)

Revision 182017-05-25 - GabrielSuarez

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Standard Polymerase Chain Reaction (PCR)

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 Final Concentrations
Template For primer Rev primer 5x Phusion buffer 10mM dNTPs Phusion poly ddH2O
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<250 ng 0.5然 0.5然 1x 200然 1.0 units/50ul PCR -
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<250 ng 0.5然 0.5然 1x 200然 1.0 units/50ul PCR -
 
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NEB recommends a template amount (for 50痞 reaction) of 50 ng - 250 ng for genomic DNA and 1 pg - 10 ng for plasmid or viral DNA.
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NEB recommends a template amount (for 50痞 reaction) of 50 ng - 250 ng for genomic DNA and 1 pg - 10 ng for plasmid or viral DNA.
 
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Although the recommended Phusion polymerase concentration is 1.0 units/50 ul rxn (20 units/ml) this concentration may have to be changed based on amplicon length and difficulty. The concentration may vary from (0.5-2 units/50 ul rxn) but should not exceed 2 units/50 ul rxn, especially for amplicons longer than 5 kb.
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Although the recommended Phusion polymerase concentration is 1.0 units/50 ul rxn (20 units/ml) this concentration may have to be changed based on amplicon length and difficulty. The concentration may vary from (0.5-2 units/50 ul rxn) but should not exceed 2 units/50 ul rxn, especially for amplicons longer than 5 kb.
 
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Thermal cycler conditions:
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Thermal cycler conditions:
  Denaturation: An initial denaturation of 30s at 98 C is sufficient for most amplicons from pure DNA templates; however, longer denaturation times can be used (up to 3 m) for templates that may require it. During thermocycling, this step should be kept to a minimum; typically, NEB recommends a 5-10 s denaturation at 98 C for most templates.
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    • Too many cells or too much residual media is a common source of failure for whole-cell PCR.
  • Add the diluted cell mixture as 1/10th the final volume of the PCR reaction.
  • Add an initial denaturing step of 10 minutes to your PCR program at 94蚓 to lyse the cells.
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Colony PCR

  • A colony from an overnight plate can be used as PCR template. Caveat: Due varying sizes of colonies, the difficulty of picking-up all cells from a colony and other factors, expect this protocol to work 80% of the time.
    • Important: Lab made phusion polymerase is NOT recommended for this protocol (it will greatly lower the chance of your PCR working)
    • Colonies to be used should have grown to a visible easy-to-pick size
    • Ideally, colonies are well spread in the plate, such to avoid cross-contamination with other colonies
  • Set a 1.7 ml Eppendorf tube (or 96-well microplate) with 250無 dH2O.
  • Have your PCR mix ready with all components - except cells - set on ice.
  • Scrape-off a whole colony from the plate - you can use a sterile pippette tip for this
  • Transfer the picked colony (all of it) into the water in the Eppendorf tube by scrapping off the cells from the pippette tip by rubbing it against the wall of the tube several times.
  • Vortex well the re-suspended colony (~10 seconds).
  • For a standard 50無 PCR reaction: add 2無 (immediately after vortexing) of the diluted colony mixture to 48無 of PCR mix in a PCR tube.
  • Add an initial denaturing step of 10 minutes to your PCR program at 98蚓 to lyse the cells.
 
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