Difference between revisions of "PCR-from purified DNA"
(→Materials Needed) |
(→Notes) |
||
| (One intermediate revision by the same user not shown) | |||
| Line 25: | Line 25: | ||
== 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 0.3 microliters of DNA in the tube by mixing with a pipette | -Suspend 0.3 microliters of DNA in the tube by mixing with a pipette | ||
| Line 34: | 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
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.
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
See Help:References for how to manage references in OMPwiki.
