I'm encountering a PCR issue in our experiments. We employ a master mix as control and utilize 3 or 4 primers in a single reaction. Since our samples are typically heterozygous (containing both wild-type and mutant alleles), it's expected to observe two bands as results. In one particular PCR, I'm consistently observing a faint wild-type band in the master mix. I've repeated the experiment using all-new reagents, but the outcome remains consistent. This light band consistently aligns with the length of the wild-type. Is it common to observe such bands depending on the choice of primers? What are the places I can improve.
It sounds like you have post pcr contamonation from a WT sample, You have fresh reagents so the contamonation could be in the water, in your pipetes or just in the dust in your working area, I would borrow pipettes,set up the pcr with 3 no template controls in another lab or at least in another researchers working area and with new plasticware and ideally a different pcr machine.While the pcr is running strip down and clean your pipettes. If eventually you cannot solve the problem then you may have to re design the wt reverse primer to give a larger product which will stop thr contamination from amplifying
Run a PCR with 2 vials each of no primer, no-template, and master mix alone. And in case you are using strips, better do it in vials so that contamination can be avoided.
It happened to me also during my initial experiment days I found during loading of wild type ,a few amount of sample was transfer to just beside well and shown as faint band in that well so you can check for this by simply loading negative controle 2 wells away from your sample well And use new NFW and if still you would get same result then most probably your primer could be contaminated as you mentioned you changed all reagents
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