I am running conventional PCR with DNA extracted from various sample types using primers (Lee & Lee, 1997) designed to identify avian pox infection. Sample types include blood, feather, tissue, plasma and RBC. Bands from feather/tissue/whole blood samples (#s 1-6 in photo) are a bit smeared (I assume due to concentration of virus in these samples/high molecular weight?) but show no non-specific primer binding or ghost bands. The plasma sample (# 7) is much fainter, but also shows no extra bands. The blank well (# 13) is clear/no obvious contamination in the PCR. "Healthy" sample from whole blood (# 14) is clear/blank as well. RBC samples (#s 8-10) however show a ton of non-specific primer binding/extra bands of varying brightness. I ran three RBC samples and all show differing patterns of extra bands. I also ran two "healthy" RBC samples (#s 11-12); these show a few extremely faint bands/smears.
Used cycling parameters from Baek et al 2020 (denature at 95, anneal at 50, extend at 72 for 40 cycles). Accustart Taq mix (pre-mixed). 4uL DNA template (per Baek et al) in 25uL reactions (tubes). 3% agarose gel with 3uL EtBr in gel and 3uL EtBr in rig (downstream). Gel ran 60V for 80 min.
Any ideas on what is going on with the RBC samples? Or any tips of optimizing this set up for virus ID/confirmation?
@Sarah Scott. From my understanding, multiple bands are due to unspecific primer binding. Recently to solve this, I increased annealing temperature, reduced number of cycles and increased mgcl conc. You can do these stepwise and also combine all three in an experiment.
Also, what is the mol weight of your anplicon? Bc if it's in thousands, 3% gel is too concerntrated.
Good luck
https://www.thermofisher.com/ma/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/pcr-education/pcr-reagents-enzymes/pcr-troubleshooting.html
hello
you can reduce amount of DNA template , reduce the cycle number and amount of agarose
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