Affect of Heating Time on DNA Fragmentation
Post date: Feb 3, 2012 9:50:02 PM
After performing a test of heated DNA (95 C for 10 minutes) versus non-heated DNA for Kristi using the ARD_SSC.C8 cell line, we found that heating DEFINITELY denatures the DNA, literally to smithereens. We decided that I should try to fluctuate the heating times between 0 and 18 minutes and compare those to non-heated DNA. I have attached an image of the gel results which turned out beautiful I am proud to say!
I used the KMS34 cell line for this experiment. The non-heated DNA stayed at the top of the gel while the other 9 samples showed quite a variety of smear patterns. As you can see, when the DNA was heated for 18 minutes, it was denatured to the point that it lies between about 500 and 100 base pairs. On the other end of the spectrum, the DNA that was heated for 2 minutes shows a really nice, long smear from about the 12000 base pair marker down to about 650 or so.
After thinking about the effects heat has to DNA, there are both pros and cons to heat denaturing, and heating to differing times. If you want to get small fragments of DNA, for instance for use in a CGH protocol, heating for 18 minutes seems to do the trick. If you want to amplify long-length DNA via PCR, I would definitely say to avoid heating 18 minutes and try to stick to a time around 1 and 4 minutes. In talking with Jonathan, this image might explain why it tends to be difficult to amplify an DNA sequence of a large size, say 8 kb for example. Because we are heating the DNA at 94 C for 5 minutes and then proceeding to heat for another 15 or so, it is no wonder that we have a hard time getting many 8 kb amplicons. They are all shredded to pieces!
Just an interesting piece of data we figured out in the Keats lab today. Ending the week with interesting results is always a treat!