I have been using universal primers to do Pcr on extracted DNA from sediment samples.
I have not changed any parameters and it used to work perfectly. I have also tried using different cyclers... what can be causing this?
The streaking might indicate that you use too much template in your PCR reaction. Dilute your samples 1/10, 1/100 and 1/1000 and try again.
Regards, Christian
Christian
I will try that thanks, my only concern is this happens to samples I've previously ran with perfect separation.
kind regards kiri
I see, you already ran these samples and it worked fine. Template DNA degradation might be another aspect. Under which conditions did you store your samples and for how long?
in a 4°c fridge ... some as long as a couple of months, but some are only a couple days old.
Freezing your samples is generally better. I used to store my template DNA samples at minus 20 degrees C. Another problem might be your oligonucleotides. If they have become degraded they will lack specificity. This might also result in the appearance of smears. Try to use fresh dilutions or use different oligonucleotides to see if the problems persist.
Good luck!
dear Kiri J Rodgers
I think your DNA sample is break. I suggest, If possible re-extraction DNA (gently) then continue.
Did you run a DNA ladder on your gel? If the DNA ladder is also smeared, it suggests the problem is with the gel and not the PCR.
If the DNA ladder looks fine, it is possible that the thermocycler is not working properly. Try using another machine.
Your dNTPs can also become degraded, particularly dATP.
I usually store my samples in 4C only if I use them the same day or next day. Otherwise it is always recommended to save them in -20C if you want to store them for longer. Also try freshly diluted primers as it is also possible that your primers are degraded. Hope you leave your primer stocks in -20C.
cycles of primer thawing and freezing may decrease their efficiency.
- Badges
- Science method