PCR-from Bacteria

You can help OMP wiki by editing the content of this page. For information about becoming a registered user and obtaining editing privileges, see Help:Accounts.

Purpose

This protocol is used to amplify copies of a desired strand of DNA isolated from a bacterial species.

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 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

References

See Help:References for how to manage references in OMPwiki.