Q5 PCR

Fill an ice bucket. Put all reagents on ice (take Q5 MM out of -30 freezer only when ready to use it, and put back ASAP). Label PCR tubes.

To make a 50uL reaction using two primers add, in this order:

Note: for gradient PCR make one large MM and aliquot into PCR tubes for efficiency. (Note: both PCR machines can do gradients, but the ProFlex one can only do two temps, whereas the Bio-Rad can do eight.)
 * Enough uL sterile H20 to make 50 total uL.
 * 2.5uL 10uM primer 1
 * 2.5uL 10uM primer 2
 * For every 100 uL reaction, add enough DNA to get 25 total ng DNA. (So, for 20 uL reaction, 5 ng DNA. Note: Q5 protocol suggests 1pg-1ng plasmid DNA in 50uL reaction.
 * 25uL Q5 MM-- and pipet up and down to MIX WELL

Vortex everything to mix before using. Thaw the Q5 by gently warming in 37℃ water bath until not quite completely thawed. Let the rest thaw at room temp (invert). Vortex to mix. Centrifuge if necessary.

Q5 program: Run for 35-45X

Note: enter the lowest and highest times for gradient PCR and the machine will automatically make intervals. Load samples in direction that temperatures are shown on screen.

Only tighten the lid until slight resistance. When PCR program is finished, press “cancel” and wait until fully cancelled to open the lid. Measure concentration. Store at 4℃.