I've tried this reaction several times over the past few weeks and tried a few trouble shooting things, but still not working. My coworker got it to work before me, I'm using his primers.
0.5uL F primer (final conc 500nM/uL)
0.5uL R primer (final conc 500nM/uL)
1uL template DNA (final amount 5ng) (I'm amplifying 3 different plasmids that have the same primers)
5uL Q5 Master Mix (NEB)
I've tried the following:
- Varying the amount of primer higher and lower than standard 500nM/uL
- Varying the amount of template plasmid to 1pg, 500pg, 1ng, 5ng, 100ng
- Re-amplifying plasmid in e coli, miniprepping to make new plasmid concentration
- Borrowed plasmid from my colleague who had successfully amplified from his tube, repeated his procedure exactly with no change
- New water
- New Q5 Master Mix tube
- New primer stock
- Longer extension time (40s/kbp) and regular extension time (25s/kbp)
- 25 cycles and also 30 cycles
- Double checked the primers match to the sequence
- Used Tm specified by NEB Tm calculator, my colleague got product using this Tm
EVERY SINGLE TIME there is NO product at all, none visible, nope. I can see my DNA ladder and also a faint primer band that varies depending on how much primer I add. When I used a large amount of template I could see a faint plasmid DNA band at the top, but there was no linearized DNA band visible.
Any insight would be greatly appreciated! I've been cloning for a few years and this is bugging me.
I was thinking of:
- Ordering new primers
But I don't have any other ideas. I would change the Tm but my coworker got product already.