I amplified a gene from Mycobacterium tuberculosis 6 months ago. I got a single band on the gel corresponding to the gene but now when I amplified the same gene with the same amplification conditions, I am getting multiple bands. I increased the annealing temperature to avoid any mispriming, but it didn't help. Positive control is working fine. Has anyone come across such an issue?
Hi, try less template. Did you check the DNA concentration before you did your PCR?
Try varying MgCl2 concentrations. Do a dilution series to determine which concentration results in the best bands.
Cheers, Nadine
Hi!
As Nadine mentioned check the conc. of DNA always once you isolate and when ever you isolate. Minimum 10 ng and MgCl2 1.5 mM is sufficient for PCR. You told ctrl. is working fine certainly problem could be with DNA conc. Try and say what happened.
Good Luck!!
Sometimes, I face this kind of problem. Try adding Glycerol or DMSO at 5% or 10% level.
You might have read what Ekemini mentioned, but you are saying that you increased Ta too still getting problem.
As Ismail n Deepak advised try using DMSO. Some of the people use additives, I got some information about them just read you get useful information.
http://www.staff.uni-mainz.de/lieb/additiva.html
http://www.biomedcentral.com/content/pdf/1756-0500-5-257.pdf
Prior to all these you should check your DNA condition that is very important..
Hi all, thank you for your suggestions, I used 25ng freshly isolated DNA and confirmed it on gel before use. I guess I will try with varying MgCl2 in addition to DMSO.
Hi all,
I used DMSO in PCR, guess what... it worked!!!!!!
The actual function of DMSO in PCR is it goes and bind to the DNA at the Cytosine residue and changes its conformation which makes the DNA more labile for heat denaturation. this is the reason for the lowering of tm and increased GC region. since most of the primers are GC rich, DMSO indirectly facilitates the annealing of primers to the template this enhaces the amplification.
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