We are working on amplifying the SSU rRNA gene of Cryptosporidium from pig fecal samples through nested PCR using the same primers, thermal profile (annealing temp 55 ℃), reagents, and machine settings that previously produced clear bands of 830 bp. However, we are now encountering issues where even our positive controls fail to show bands.
Despite trying multiple adjustments—including changing annealing and denaturation temperatures, using different enzymes and thermal cyclers, using new set of same primers, re-extracting DNA from the stock samples to rule out degradation, and testing various sets of positive controls—we still aren’t obtaining bands. Two days ago, we managed to see a faint band of one sample (first picture, well no. 5), but no bands have appeared ever since (2nd picture).
Has anyone experienced a similar issue or have any suggestions on troubleshooting this problem?
Hmm, have you tried getting fresh dNTPs? They weren't mentioned above so it's worth a try. They do expire or degrade, especially if they have been frozen and thawed multiple times. Any chance of a nice plasmid to amplify from for a positive control?
Hope this helps and good luck!
When you say "fresh set of same primers" do you mean you made a new dilution from the stock tube in the freezer or do you mean you ordered a newly synthesized new tube of the same primers?
Primers can degrade, even frozen stock solutions.
Also, I know it sounds silly, but double-check the thermocycler program to make sure it doesn't have an error. I've had someone else accidentally edit my thermocycler programs & not tell me.
Good luck!
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