Skatebro:PCR

Skatebro:Project08

Tips

• Want about 50μL volume in each tube, 1 ng template DNA, 300nM of each primer (~200ng)
• Try running at 55c regardless of melt temps from primer design programs
• If no binding on first run, try a temp gradient trial from 48-64c
• Do a pcr clean up (elute down with 30 microL instead of more) and run a gel with control fragment similar in size
• Try running PCR reaction with betane for low GC sequences

Steps

1. Dilute plasmid/template DNA to a concentration of 1ng/μL, and label this tube 'XXXX for PCR'
   • Dilution review: If starting concentration of plasmid DNA = 50ng/μL, add to a tube 49μL of water and 1μL of the 50ng/μL plasmid DNA.
2. Prepare 25μM tubes of each primer (if you don't have them already)
3. Add in order: (total reaction volume of 50μL)
   • 49 μL of Tom's PCR mix
   • 1ng template DNA
   • .6μL of each primer (forward and reverse) (.6 μL of the 25μM primer yields roughly [300 nM])
4. Run the tubes on the thermocycler at an appropriately designed program:
   • ~95deg for 3:00
   • 30 cycles of (a) melt (b) anneal (c) extend
   • 72deg for 10:00