This is my PCR 1.2% agarose gel result. I used a 25uL reaction system (0.3uM primers, 12.5uL Taq master mix, 1uL genome template and 5.5uL H2O). The primer Tm is 62℃, and reaction procedure is 94℃ 3min--(94℃ 30sec--57℃ 30sec--72℃1min)25cycles--16℃. What is the possible reason for the existence of the bottom band around 100-250bp? How to optimize my PCR procedure? Thanks!
Are you sure that this is a pcr artifact?. You do not say exactly how much dna you are adding in 1ul. try running a dna sample diluted to the same volume as the pcr to check that the low size smear is not just degraded dna in which case you are using to much dna in your pcr and using less may give stronger large bands if your dna contains pcr inhibitors
Hi Paul, Hi Anita
it seems that PCR amplicons are not at same size among samples. is it true?
in addition to degraded DNA, you should also see smears above PCR bands, you can have excess of primers and also multimerizations of primers.
primers are always in excess, and overall PCR must be standardized with right conditions, right amount of each goods...you could try using less amount of primers, just to see.
all the best
fred
You do not specify how much DNA, if any, is being added to the mixture in 1ul. It appears that there is a size discrepancy in PCR amplicons between samples. Overlapping bands, primer excess, and primer multimerizations are all possible causes of smears above PCR bands. Altering the DNA and primer concentrations may help.
Hello Anita,
maybe your annealing temperture is too low with 57°C. Increase it to 59°C, 61°C, 64°C. Have you made a gradient PCR to determine which annealing temperature is the best? When the primer binds more specific to the target then the smear bands of primer artifacts will disapear automatically.
Have you checked if your forward and reverse primer cross dimerize at their 3´ end because then the polymerase can extend them to 70 bp artifacts.
Best tool is Netprimer (PREMIER Biosoft: User Login), make an acount and then copy your forward and reverse primer into the respective lines and set the "Temperature for Free Energy Calculation" from 25 to 60°C and analyze. Then you know how good is your pair, free energy should be smaller - 8.0 and no cross-dimers at best.
Genomic DNA should be used with 50 ng/reaction, that is most time sufficient, otherwise, as the other three mentioned, the DNA degrades or even inhibits your PCR to a certain extend.
Best wished Volker
Usually PCR products run on 2% gel. If it is RAPD use 1.5% Agarose gel. If you want to reduce non specificity increase Tm so that extra bands might reduce. If you have Gradient facility you can check for range of Tms. It is appearing like a smear not band. Try once again carefully. Good Luck!!