Actually, I am doing pcr for Kleb.pneumonia gene. I have did pcr of 90 samples initially and all were positive at that time. But now again when i am repeating the pcr for those 9 samples with same pcr conditions, same conc of dna and same pcr mix, results are varying. only few samples are now showing positive.
Can anyone tel me the problem behind this variations ???
What do your controls look like?
My guess is that your DNA samples, or primers, or both have degraded.
If your primers degraded, then making a fresh batch will solve the problem.
If your DNA samples have degraded, there really isn't any way to fix the issue.
Dear Sneha Bhowmick which PCR did you do? TaqMan or SYBR green based real time? Or classical PCR?
Haw many samples did you analyze for the first time and now? 9 vs 90...
It was expected data - all positive? It were clinical samples or controls, culture etc....
Please give me more information.
Yes I have touched with time-depended problem of reproducibility. But I cant understand your start conditions.
What do your controls look like?
My guess is that your DNA samples, or primers, or both have degraded.
If your primers degraded, then making a fresh batch will solve the problem.
If your DNA samples have degraded, there really isn't any way to fix the issue.
You can Cross check some freshly isolated DNA samples , seems some inhibitor presence
If this is end point pcr then I agree with Katie A Burnette that primers may be a problem and that controls are essential. Also in pcr very small differences in pcr efficiency will cause big differences in the yield so if your early pcr was on the edge of working and then you move to a different pcr machine, different cycle or even different plates/thickness of tubes then some weak positives may become negative when viewed in a gel
Andrei S. Babenka it was a classical PCR ..and at first i did PCR for 90 culture samples and when again i repeated the same DNA samples after 10 months with same 90 samples .. results were not same.. now few samples showed positive for k.pneumonia compared to earlier results.
ok. Sneha Bhowmick Did you measure DNA concentration prior of the first PCR? Could you check it again now for all samples? Please use fluorimetry.
May be increasing of start DNA concentration can help, if we think that it is DNA degradation.
Moreover, we can check DNA fragmentation rate with Agilent Tape Station or Qiagen Qiaxcel - capillary electrophoresis. It could provide you an important information.
I think that it could help. Try it if possible.
Hi, Sneha Bhowmick, if your DNA samples were at -80 C, should be ok, then the problem was the primers, making new stock should solve the problem... however, if your sample were not at -80 C the DNA probably is degraded.
If your DNA sample is saved in proper condition and not corrupted, it will gave you same result 👍
I suggest you to repeat the PCR for a few randomly selected samples to make sure that everything was fine with your second PCR, while you are doing the things suggested above. Pay more attention to the PCR water, pipettes etc. that you used to prepare the master mix.
The concentration of DNA drops approximately 10 times during a year of storage. This way you will never get the same PCR results after 9 months with the same reagents.
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