PCR-from purified DNA

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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

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References

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