Difference between revisions of "PCR-from purified DNA"
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-Add to a 0.2 mL PCR tube 10 microliters of LongAmp Taq Reaction Buffer, 1.5 microliters of 10 mM dNTPs, 2 microliters of 10 micromolar forward primer, 2 microliters of 10 micromolar reverse primer, 2 microliters of LongAmpTaq DNA Polymerase and 32.5 microliters of nuclease free water | -Add to a 0.2 mL PCR tube 10 microliters of LongAmp Taq Reaction Buffer, 1.5 microliters of 10 mM dNTPs, 2 microliters of 10 micromolar forward primer, 2 microliters of 10 micromolar reverse primer, 2 microliters of LongAmpTaq DNA Polymerase and 32.5 microliters of nuclease free water | ||
| − | -Suspend | + | -Suspend 0.3 microliters of DNA in the tube by mixing with a pipette |
-Set the PCR machine to have an initial denaturation at 94 C for 30 seconds, 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 | -Set the PCR machine to have an initial denaturation at 94 C for 30 seconds, 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 | ||
Revision as of 11:54, 13 July 2015
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Contents
Purpose
This protocol is used to amplify copies of a desired strand of DNA isolated from purified DNA.
Materials Needed
-PCR strip of microtubes
-LongAmp Taq Reaction Buffer
-10 mM dNTPs
-10 micromolar of appropriate forward primer
-10 micromolar of appropriate reverse primer
-LongAmpTaq DNA Polymerase
-Nuclease free water
-DNA template
-PCR machine
-micropipette
Protocol
-Add to a 0.2 mL PCR tube 10 microliters of LongAmp Taq Reaction Buffer, 1.5 microliters of 10 mM dNTPs, 2 microliters of 10 micromolar forward primer, 2 microliters of 10 micromolar reverse primer, 2 microliters of LongAmpTaq DNA Polymerase and 32.5 microliters of nuclease free water
-Suspend 0.3 microliters of DNA in the tube by mixing with a pipette
-Set the PCR machine to have an initial denaturation at 94 C for 30 seconds, 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
Related Ontology Terms
References
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