purification of long single stranded DNA - (Nov/09/2005 )
I am not a biologist or biochemist and wonder if I cold get help with the following from an expert:
I am trying to purify long (around 1 micrometer say) single stranded DNA, the aim is to get rid of shorter polimerization products primers and nucleotides. It seems using a low percentage agarose gel is the best way to do that. It is important for me however to get back intact DNA, i.e. it mustn't rupture. Double stranded DNA seems quite resilient to mechanical strain and other things that might cause rupture. But how sensitive is single stranded DNA? What are the things one shouldn't do, pipetting, vortexing, using qiagen yellow buffer, using the qiagen columns from the PCR purification kit, what is bad for single stranded DNA?
Thanks a lot
1 micron is pretty short as such things go. I'd recommend a sepharose G-50 or G-25 gel filtration column to get rid of small molecules. Another option would be a Millipore Microcon centrifugal filter. Don't vortex or use narrow tip pipet tips. Wide bore pipet tips are available, or you can cut the tips off normal ones.
this answer may seem quite laughy... but i did suppose that acrylamide gels were more precise than agarose gels...
as phage434 said, vortexing and pipetting up and down is quite forbidden. I'm not too sure, microcon centrifugal filter have a risk to break the DNA too. I would recommend a sepharose column too.
Thanks both of you.
Sepharose G-50 or Sephadex G-50?
From Amersham right?
On the webpage it says that the exclusion limits are of the order 27 bases for sephadex G-100, the largest available. My primers are much longer than that, over 50 bases. Also I would like to get rid of things that are say 200, or 300 or so bases long etc., ideally I just get back DNA in a very narrow size range. I have already tried bio rad p 30 bio spin colums and the qiagen columns from the pcr purification kit. Both leave the long primer in the DNA solution unfortunately. Are they any worse than the Amersham colums?
Agarose seems to give me kind of a very slow band and it has turned out to contain long DNA. Primers are filtered out. Acrylamide? I have tried, but I need such a low percentage, that the gel would just rip apart. I can't go below 5 % say and at that percentage, the large DNA hardly enters the gel. Maybe leave it running for a couple of hours?
I will be more carful with pipetting, will cut off the tips as you suggest. If you suggest to be careful even with up and down pipetting, then single stranded DNA must be in fact really sensitive.
in this case, centricon columns may help more...
you can find infos on the manual here :