One way to mitigate primer dimer formation is to reduce nCoV assay primer conc. Let's test 800, 400, 200, 100, 150, 50 nM for both N1 and S2 assay. Based on gradient results, we'll just do two plates at fixed temps, 60 and 68 deg. We'll add spike at 1e4, 1e3, 1e2, 1e1 x3, 0 x 2 to get a nice template range and bias towards 10 copy condition.

NOTE: I messed up and added 2x these primer concs, so we were actually at 1600, 800, 400, 300, 200, 100.

One other change— from now on we're doing 20 mcL PCRs to sample more material.

• rxn mix:
  • 10 mcL Luna MM
  • 1 mcL Luna RT
  • 2 mcL _ mcM N1 or S2 primers with P5/P7 adapters (16, 8, 4, 3, 2, 1 mcM)
  • 7 mcL HEK293 lysate (1:100 diluted in 0.5X heat-inactivated lysis buffer [diluted 1:1 with water])
  • 18 total without primers *8=144 mcL
• master mix:
  • 10 MM *8=80 *25=2000
  • 1 RT *8=4 *25=200
  • 2 primer mix *8=16
  • 7 lysate *8=28 *25=1400

spike in 1e4, 1e3, 1e2, 1e1 in triplicate, 0 in duplicate copies of Twist RNA at each primer conc.

Results

400 nM seems like our best bet at 60 deg:

drilling down on the 400 nM condition, the HKU primers look really promising!

Ran gel on 60 deg 400 nM primer conditions for N1 assay. Expect to see ~130bp bands:

1. 1e4 Twist RNA copies
2. 1e3
3. 1e2
4. 1e1
5. 1e1
6. 1e1
7. 0
8. 0