Hello,
I am attempting to amplify two fragments of Tobacco Plastid DNA, each of a length of 1.2 kbp. The PCR worked once, two months ago but I have since needed to replicate for further work but now it will not work. The primers were designed with NCBI Primer BLAST, and in addition I ordered primers to the 23 and 16S ribosomal subunits as positive controls. I have tried re-extracting template DNA and making fresh aliquots of primers yet no result.
-Standard CTAB extraction with chlorofom/isoamyl purification with either isopropanol purification or silica column purification
-Phusion polymerase with associated buffers/dNTP etc.
-Varied TM's and extension times from 65-45 C
-Recent, yet failed, addition of a positive control consisting of 23/16 S RNA subunits.
I am stumped. The only place I can think this is going wrong is my template. I know the primers worked from the past plus the conserved nature of the subunits. I also know my reagents work from other successful PCRs. Is there something I should change in my extraction to perhaps get more and higher quality plastid DNA?
Any help would be greatly appreciated,
Thanks
Can you go back to original stock primers? If the primers were dissolved in water not TE then they may have depurinated or been degraded by nucleases in the water. Try ordering new primers and dissolving in TE.
The failure of the positive control means there is something globally wrong & not unique the the plastid DNA samples.
Double-check the thermocycler settings. If you use shared equipment, someone may have changed the program.
Get a new tube of enzyme. If it has a separate buffer, make sure the Magnesium hasn't precipitated.
And order new primers.
Good luck!
Thank y’all for the responses, I will try them out and post if any of the suggest changes in conditions/reagents solves the problem.
Cooper A. Svajda
Did you positive control work? Also, check your PCR machine. In our lab, after a few years of running, a couple of our PCR machine machine broke down, and needed maintenance.
Hey y’all,
So I did a complete revamp. I re-extracted DNA with an emphasis on removing nuclei with centrifugal fractionation. I made new working stocks of primers. I diluted the DNA 10-fold and ran it at 40 cycles. This combo of steps gave me my bands. Thank you all for your insightful answers!
Well done Cooper A. Svajda it is always good to hear that things are working
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