I have performed agarose gel electrophoresis 1,2% for the PCR product. My primer set was designed to yield 750 bp fragment. But, on the gel after PCR, I saw a band below 200 bp (below the 13th band of DNA ladder). So, what do you think of the cause? Should I increase the annealing temperature or maybe there's an error when I made the master mix so it's the primer that showed up on the gel?
Dear Yessica...
You have two different issues, the below 200bp band and the absent targeted band. In general, we have doubts about bands with low molecular size to be primer dimer, and negative control will check this out!! The absence of your specific band may be results from primer failure or annealing temperature! You have to check them both!
Best Luck.
Dear Yessica...
You have two different issues, the below 200bp band and the absent targeted band. In general, we have doubts about bands with low molecular size to be primer dimer, and negative control will check this out!! The absence of your specific band may be results from primer failure or annealing temperature! You have to check them both!
Best Luck.
That might be because of non specific primers, low concentration of the template, non specific annealing temp, or not good master mix (e.g. DNA polymerase, dNTPS, Mg..etc). The lower band is more likely primer dimer. Good luck
Hi
I think in error master mix
if possible Upload a picture
thanks
I agree with Mushtak...
Re-check the primer specificity! There is a big chance that the band with low molecular size is dimer!
Best luck.
Looks like a dimer. You need to check the primer's specificity. And if you are sure that primer are specific then you need to play with the annealing temperature. Sometimes, poor template quality is also a reason of PCR failure. Also check the reagents as multiple freeze-thaw cycles degrade the reagents efficiency. Good Luck
Hello Yessika,
This looks like a primer dimer. What you can do is, 1) run a primer BLAST to see if there are any non-specific bindings, 2) using a bioinformatics tool to see the formation of dimers or secondary structures between primers.
the band which you saw on gel might be a primer dimer. you need to optimize your primer concentration and annealing temperature for your primer pair
Hi Yessica
This is definitely a primer dimer. You ought to optimize the annealing temperature for your primers. Running a gradient PCR could help you get the right annealing temperature. All the best.
Hello Yessica, it looks like a primer dimer, note that the band is faint. These type of bands below the ladder, usually happen when you have primer dimer or an excess of primer. In your case these excess of primer could happened because your PCR did not work. You should check your master mix, for example, some Taqs already have Mg in their buffer, other ones you need to supplement it yourself. If your master mix is ok, you should try a gradient with several annealing temperatures. Normally when you have unspecific bands you should increase annealing temperature and when you lack amplification, you should decrease temperature. Of course check your primers and if it doesn't work anyways, maybe you should design another set of primers! Good luck!
- Badges
- Science method