PCR – Repeat of RA Primers on LCM and Genomiphi DNA

Due to the failure of two consecutive attempts, I am repeating this PCR using the reagents and cycling params listed in Lisa’s notebook. Master mix calcs are here. Not noted in the master mix calcs are the MgCl2 and dNTP stock concentrations. They were 25mM and 10mM, respectively.

Cycling params:

  • 95C – 3mins

40 cycles of:

  • 95C – 1m
  • 62C – 30s
  • 72C – 30s
  • 72C – 10m

Positive Control: “1:100 Plasmid RLP” from 7/24/09. This was supplied by Lisa to use as a positive control for these primers.

Results:

Lane 1 – Hyperladder IV

Lane 2 – LCM Classic

Lane 3 – LCM New

Lane 4 – Genomiphi Classic

Lane 5 – Genomiphi New

Lane 6 – Positive Control

Lane 7 – NTC

It worked, finally! Both LCM samples clearly amplify. However, neither of the Genomiphi prepped samples produce any product. Will discuss with Lisa and/or Carolyn to see what they think about this and what they’ve seen before. But, it seems clear that the Genomiphi procedure did not work. Could be due to age of the kit (see 20110414 for notes on expiration date)