How does gel electorophoresis show the degradation of the DNA and in which form does the degraded DNA appear?
If the DNA is degraded you will get a smeared band (But smear bands may also appear due to High salt concentration and if we load more amount of sample). Therefore always run standards (ladder) to compare the results. Many a times you could also observe protein contamination, the precipitated materials usually doesn't move much from the wells. This could be avoided by use of proteinase K. However, there are number of precautions to be taken care of; which may lead to DNA degradation. Please find attached Troubleshooting Guide for DNA Electrophoresis for your kind perusal. Hope it will be useful for you. All the best :-)
If the DNA is degraded you will get a smeared band (But smear bands may also appear due to High salt concentration and if we load more amount of sample). Therefore always run standards (ladder) to compare the results. Many a times you could also observe protein contamination, the precipitated materials usually doesn't move much from the wells. This could be avoided by use of proteinase K. However, there are number of precautions to be taken care of; which may lead to DNA degradation. Please find attached Troubleshooting Guide for DNA Electrophoresis for your kind perusal. Hope it will be useful for you. All the best :-)
smear only appears if degradation is extensive. Indeed, due to the double helix structure, single strand cuts may not appear. So, in order to really check for degradation you need to denature dna sample before running the gel.
An example is available on request.
Hi,
My problem is that the DNA shows a ratio of 1.9 in 260:280 using nanodrop and the DNA in gel appears good too.But amplification in PCR doesnt happen (control works for the regions)....any suggestions????
If the DNA looks reasonably intact by gel electrophoresis then it likely will be fine as a PCR template. I would look elsewhere for the problem, for example maybe your primers are not good, or you are adding too much template, or your PCR conditions are not suitable, or the DNA is contaminated with a PCR inhibitor.
Where is your DNA from? Environmental samples can contain high levels of humic substances which can inhibit your PCR. Do you have a positive control? If it is environmental then a 1:10 dilution is good. Also, DMSO can work wonders in a PCR. I have set up a few PCRs that won't work without it.
Thank you Michael..But my primers works well for other DNA samples which I process. I also checked the sequences of those amplified samples.The protocol is already optimised. With ref to the DNA template, I have tried dilutions too... but PCR inhibitors!!!!!!! how would i get rid of them????
Thanks Ian ...I have optimised the protocol using a positive control without DMSO...My samples are DNA from human peripheral blood...I have the problem with only one sample.... while all other DNA works for this optimised protocol....while the quality of DNA in gel proves good...i wonder where things go wrong...
I would check with a different set of control primers on the problematic samples to see if those work. Otherwise can you just clean up the DNA by ethanol precipation?
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