Difference between revisions of "PCR-from purified DNA"
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== Materials Needed == | == 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(purified DNA) | ||
| + | |||
| + | -PCR machine | ||
| + | |||
| + | -micropipette | ||
== Protocol == | == Protocol == | ||
| − | -Add to a 0.2 mL PCR tube | + | -Add to a 0.2 mL PCR tube 5 microliters of LongAmp Taq Reaction Buffer, 0.75 microliters of 10 mM dNTPs, 1 microliter of 10 micromolar forward primer, 1 microliter of 10 micromolar reverse primer, 1 microliter of LongAmpTaq DNA Polymerase and 16.25 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 | ||
| Line 15: | Line 34: | ||
== Notes == | == Notes == | ||
| + | The varying times for the steps will depend on the conditions for the primer you are using | ||
== Papers where this or a similar method has been used == | == Papers where this or a similar method has been used == | ||
Latest revision as of 13:59, 20 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(purified DNA)
-PCR machine
-micropipette
Protocol
-Add to a 0.2 mL PCR tube 5 microliters of LongAmp Taq Reaction Buffer, 0.75 microliters of 10 mM dNTPs, 1 microliter of 10 micromolar forward primer, 1 microliter of 10 micromolar reverse primer, 1 microliter of LongAmpTaq DNA Polymerase and 16.25 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
The varying times for the steps will depend on the conditions for the primer you are using
Papers where this or a similar method has been used
Related Ontology Terms
References
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