Streptococcus Transformation - NSJ Amplification

For sixteen 10ul reactions (making enough for seventeen): PCR Recipe:

• • 96.9ul Molecular grade water
  • 34ul 5x Q5 Reaction Buffer
  • 3.4ul 10mM dNTPs
  • 17ul Primer mix (1:20 dilution of IDT O-276 and O-277 primers)
  • 17ul DNA template (1:20 dilution of relevant genomic DNA)
  • 1.7ul Q5 polymerase

Quickly vortex and aliquot into 10ul reactions.

PCR Machine Protocol:

• • 98° for 30 seconds
  • 98° for 10 seconds (Denaturing temperature)
  • 53° for 30 seconds (Annealing Temperature)
  • 72° for 2 minutes (Extension temperature; Use 30 seconds per 1kb of expected product)
  • Go to step 2, and repeat 34 times (so, 35 cycles total)
  • 72° for 5 minutes (Allow polymerase to synthesize any unfinished strands)
  • 12° hold

Combine all reactions; then, run 2ul out on a gel to check if this procedure worked. The expected product size is 2.7kb.