We are optimising primer for PCR,whose length is 20 bp,Tm 59.4,GC content 55.We have got bands,but we couldn't visualise it properly in Agarose superior grade type II(1.2% gel).I have attached my gel picture. can we confirm them as PCR bands or not ? could anyone guide us.
Hi there,
You should include a DNA ladder : it will allow to roughly calculate the size of the band and to it will show if the detection is efficient (as you know how much material is in each DNA band)... I would suggest to repeat the running on the gel and to let it migrate further (your band is too close from the well to evaluate its size.
At this stage, one can't say if it's a detection problem (low signal), a PCR problem (low yield) or both.
What size is your PCR product? How much genomic DNA did you use as input in your PCR reaction?
For me it looks like that there is no amplification product but your input genomic DNA is visible, however, without DNA ladder, as mentioned by Dominique, this is impossible to judge.
Well, how long is your PCR product band? The band is way up there, look like a very huge size band (if you have run your gel for a while). The huge DNA band could be also due to your adding too much DNA template (gDNA or plasmid). In that case, you don't have a PCR band. It should be a good practice to reserve a lane for DNA markers.
As mentioned, do add a ladder. Run your gel slightly longer (looks like a larger amplicon) and perform a gradient PCR (vary temperatures) to dermine optimal annealing.
As already mentioned by others, include a DNA ladder ... Do you know the size of the amplified product? I am also agree with Dr. Yau that its looks like a very huge size band (just an assumption). Run the gel again: Agarose 1.5%, 50-60V, 2-3hrs., include a lane for DNA ladder (mostly the Lane 1).
Also add to your gel a negative control (PCR mix + H2O).
For the next PCR attempt, use annealing temp. at 55C.
Dear Pratyusha Kasibhotla,
Running a ladder will be good to locate your required band.
Yes, a ladder is important as it will make it easier for you to visualise your target bands.
Good luck
I think DNA ladder is necessary and it seems your product has not been run enough. and it could be because of the gel concentration(1.5%) .As others mentioned above, negative and positive control is required to see if the problem is because of your primer for example maybe TM is not correct! or is it due to running buffer or other stuffs.
good luck
Hi everyone, Thank you all for your suggestions. As suggested we added DNA ladder through which we could know that no amplification was done and those bands observed were DNA bands. Now we trying out optimization by adjusting the denaturation time/temperatures.
Pratyusha, Thanks for updating. Check the primers first to see if they are correct in content and in orientation. Then, working on the Tm. If those bands on the picture is template DNA (as you stated), that's a lot of template DNA you added in a PCR reaction. Try to decrease the amount of template DNA you add next time. Some people even dilute the DNA sample before use for PCR to minimize the possibility of PCR inhibition.
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