We are linearizing a vector with a HF polymerase (Q5 from NEB and its recommended parameters). I established conditions that produced a sharp and unique amplicon, but I've just tried it one more time and all that could be seen is a uniform smear and a faint band (were it belongs). I'm speculating that primers are degraded but why the smear? I dismiss insufficient template because I'm already including 10 ng of pDNA for a 50 ul reaction.
Thanks my friends
Hello Juan,
similar smears can be observed when you re-ampify a PCR product. Primer degradation is also certainly possible. These degraded oligonucleotides can bind non-specifically to your template so that a population of differently sized amplicons is produced (this is the smear when you separate the sample electrophoretically and stain it subsequently). This is even more pronounced if you have some residual genomic DNA in your preparation. Even tiny amounts of bacterial DNA (contaminant after isolating your plasmid DNA) are sufficient. Use fresh oligonucleotides. If this does not help I would suspect a problem with your template DNA. Good luck with your experiments!
Regards, Christian
it is possible that you have post pcr product contamination of one of your reagents and this is over amplification. Are you running a negative control sample and does it produce the same amplification effect
Thank you, Christian, for the insight on the possibility of micro-annealing. Paul, negative control was indeed performed, it corresponds to the lane in the left. Thank you, I will start with new miniprep and primer dilutions, and alternative reagents if possible.
lovely clean control Juan Pablo no hint of contamination. Go with Christian's suggestion. One thing about primer degradation. I believe that primers dissolved in water degrade by depurination as the water absorbs CO2 from the air and becomes acidic. Dissolve the primers in alkaline TE to minimise degradation both by nucleases and depurination. Good luck.Paul
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