Calculating concentrations (Qubit) and molecule numbers:
starting primer = 30 ng in 300 uL in 1 ng/uL, added 100 uL = 100 ng
100 uM = 100E-12 mols/uL add 100 uL, = 10 nmols starting primer.
Y PCR: 73.7 ng/mL = 14.7 ug/mL. in 9 mL = 132 ug dsDNA Y-library from PCR
ug/ (N* 660 g/mol)10^6 = pmol 132/(116660)*10^6 = 1.7 nmol of Y-library from PCR. (expected ~10 nmol!!)
B PCR: 51 ng/mL = 10.3 ug/mL. in 9 mL = 92.7 ug dsDNA B-library from PCR
ug/ (N* 660 g/mol)10^6 = pmol 92.7/(116660)*10^6 = 1.2 nmol of B-library from PCR
85 ug/mL after EtOH, recovered in 700 uL = 59 ug dsDNA after elution 63 ug/mL = 44 ug dsDNA after elution
Both Preps had similar loss:
80-90% lost in PCR why?
50% lost in precipitation and resuspension!! (Let’s go back to the small tube EtOH extraction)
To troubleshoot PCR:
- Try series of 1 mL PCRs
- Try more library?
- Try more nucleotides? – make fresh aliquots at 10 mL scale.
- Try more primer?
- bad PCR temperature conditions?