PCR-from Bacteria: Difference between revisions
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== Protocol == | == Protocol == | ||
-Add to a 0.2 mL PCR tube | -Add to a 0.2 mL PCR tube 10 microliters of LongAmp Taq Reaction Buffer, 1.5 microliters of 10 minimolar dNTPs, 2 microliters of 10 minimolar forward primer, 2 microliters of 10 minimolar reverse primer, 2 microliters of LongAmpTaq DNA Polymerase and 32.5 microliters of nuclease free water | ||
-Suspend one colony of the bacteria in the tube by mixing with a pipette | |||
-Set the PCR machine to have an initial denaturation at 94 C for 5 minutes, 30 cycles of 94 C for 10-30 seconds, 45-65 C for 15-60 seconds and 65 C for 50 seconds per kilobase pair of the DNA fragment, then 65 C for 10 minutes for the final extension, and hold at 4-10 C | |||
-Run the PCR machine with the tube inside | |||
== Notes == | == Notes == | ||
Revision as of 18:24, 9 July 2015
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Purpose
This protocol is used to amplify copies of a desired strand of DNA isolated from a bacterial species.
Materials Needed
Protocol
-Add to a 0.2 mL PCR tube 10 microliters of LongAmp Taq Reaction Buffer, 1.5 microliters of 10 minimolar dNTPs, 2 microliters of 10 minimolar forward primer, 2 microliters of 10 minimolar reverse primer, 2 microliters of LongAmpTaq DNA Polymerase and 32.5 microliters of nuclease free water
-Suspend one colony of the bacteria in the tube by mixing with a pipette
-Set the PCR machine to have an initial denaturation at 94 C for 5 minutes, 30 cycles of 94 C for 10-30 seconds, 45-65 C for 15-60 seconds and 65 C for 50 seconds per kilobase pair of the DNA fragment, then 65 C for 10 minutes for the final extension, and hold at 4-10 C
-Run the PCR machine with the tube inside
Notes
Papers where this or a similar method has been used
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Related Ontology Terms
References
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