PCR

From FreeBio

{Note: The following protocol uses Platinum Taq polymerase to perform polymerase chain reaction (PCR). A nice diagram that explains PCR can be found at http://members.aol.com/BearFlag45/Biology1A/LectureNotes/lec24.html .}

1. In a 0.5ml microfuge tube (which fits the PCR machines), mix:
   {Note: Remember to use a master mix without the DNA (and primers, as appropriate) for multiple reactions.}
   • 5ul 10x PCR buffer
   • 1ul 10mM dNTP mix
   • 2ul 50mM MgSO₄
   • 1ul 10uM primer 1
   • 1ul 10uM primer 2
   • 1-4ul 1:500 template DNA
     {Note: As negative control, use ddH₂O instead.}
   • 0.2ul Platinum Taq polymerase
     {Note: This volume is for 1.0 unit. Up to 2.5 units can be safely used in a 50ul reaction mixture.}
2. Add ddH₂O to tube for a total of 50ul.
3. Briefly spin down in a picofuge.
4. Program the PCR machine with the following general guidelines:
   (Note: Remember that annealing temperatures, and annealing and extension times should be adjusted depending on the lengths and G+C contents of the primers used.}
   1. 94C for 10min.
   2. 94C for 30s to melt templates.
   3. 50C for 30s to anneal primers.
      {If GC content > 50%, anneal at 60°C.} --dna1112 16:47, 6 Jul 2005 (EDT)
   4. 72C for 2min to extend primers.
      {Or ~1min/kb extension} --dna1112 16:47, 6 Jul 2005 (EDT)
   5. Return to step 2 for 29 additional cycles.
   6. 72C for 10min.
   7. 4C for as long as needed until reactions are retrieved.
5. Store short term @ 4C, and long term @ -20C.

Links

• http://info.med.yale.edu/genetics/ward/tavi/Guide.html