phage DNA - DNA wont migrate on agarose gel (Nov/17/2010 )
I have extracted phage DNA using the following protocol: ZnCl2 phage precipitation, guanidine thiocyanate lysis buffer, phenol chloroform extraction and ethanol precipitation. The concentration of DNA when read on nano drop is ~1000ng/ul and forms a nice peak at 260nm and a dip at 230. When I run the sample on a 0.7% agarose gel at 90V for up to two hours the DNA can be seen fluorescing from the well but will not migrate. I digested it with DNase so there is definately DNA there. I believe it could be 200kb but at this size it should still migrate somewhat in such a low precentage gel. Can anybody suggest a reason why the DNA will not migrate? Any suggestions would be much appreciated.
A puzzled post grad.
From the image you uploaded, I would say your phage DNA is there. 200 kb is a lot. I ran my 40 kb phage DNA on a 0.4% gel overnight at very low voltage. Less than 0.4% is very difficult to handle, but 0.4% is possible. Otherwise DNA of this size is run on a pulsed field electrophoresis. Why do you need to run it on a gel anyway?
BioMiha on Wed Nov 17 18:38:43 2010 said:
From the image you uploaded, I would say your phage DNA is there. 200 kb is a lot. I ran my 40 kb phage DNA on a 0.4% gel overnight at very low voltage. Less than 0.4% is very difficult to handle, but 0.4% is possible. Otherwise DNA of this size is run on a pulsed field electrophoresis. Why do you need to run it on a gel anyway?
Hello Biomiha,
Thanks for this feed back. What you say is reassuring and it is what I thought when I sent my DNA for sequencing. However, the sequencing company returned to me saying they thought it was contaminated because it was stuck in the well. They carried out a phenol chloroform purification step on it and the concentration droped from 550ng/ul to 85ng/ul. Unless they preformed multiple phenol chloroform extractions should such a purification step reduce the concentration by so much?
I shall try pulse field gel electrophoresis next week.
What do you think of the idea of, using rapid/universal primers to try amplify parts of the phage DNA. If it can amplify a pcr product the DNA should be accessible for sequencing, right?
Thanks again for your helpfulness
Rapid primers could do the trick if you just need to confirm, that your DNA is phage DNA. I would not count on amplifying any specific region. I am not really sure, what exactly you are trying to do. If you explained a bit, I could help more.