3 replies to this topic

[keary]

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.

Attached Files

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Edited by keary, 17 November 2010 - 08:39 AM.

[BioMiha]

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?

[keary]

BioMiha, on 17 November 2010 - 10:38 AM, 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

[BioMiha]

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.