Can anyone help with why my PCR worked one day and not the next?
I am attempting to PCR amplify my empty vector backbone ahead of Gibson Assembly and am using one backbone with two sets of primers. I ran small test PCRs (20ul) under various conditions for optimisation (Gel 1- test) using Phusion Pol and the NEB-suggested annealing temp and Tm. The conditions shown in lane 1 and 2 worked beautifully and show amplification and slight weight change from the input ev (lane "In") as expected.
The DNA template was kept at 4oC for a couple of days before I then repeated the PCR at a larger scale (50ul) to make enough template for my Gibsons. I used exactly the same conditions, DNA template, buffers, dNTP, polymerase alliquot etc with fresh sterile pure water, and even used the same saved PCR programme on the same thermocycler. And I just got smears! (Gel #2, lanes 1/2). I have since used fresh alliquots of everything, including re-dilution my template DNA and primer stocks in case there was something in the water, but I still consistently get smears.
Can anyone help troubleshoot why my results have changed from Gel 1 to Gel 2?
Hi Elizabeth,
You must be aware that there are many parameters that come into play while standardizing a PCR reaction. Since you have got the desired product amplification I would primarily ask to repeat the same conditions without the scale up. It seems like you are preparing everything (DNA template, buffers, dNTP, polymerase aliquot etc) fresh prior to your experiment so contamination can be ruled out at the moment.
If you are unable to get reproducible results with 20ul reactions (like you previously did) then we can take into consideration individual PCR components including the reaction vials and gel preparation to trouble shoot the problem.
Good luck with your experiment!
Dear Elizabeth Radley
It is probably because of:
- Badges
- Science method