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?
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