Hello,
I have been using Polyethylene Imine (PEI) on a regular to precipitate DNA from crude bacterial extracts in order to get rid of the DNA on my way to purify proteins of interest.
I am currently interested in using the PEI precipitation as a quick and dirty efficient yet cheap procedure for isolation of the nucleic acids themselves from the crude extract. The only problem is how to get rid of the PEI after capturing the nucleic acids and precipitating the PEI-nucleic acid complexes.
Can anyone here recommend a quick yet efficient approach for the clearance of PEI so that I can purify the nucleic acids captured in the PEI precipitation step? Maybe a change in solution conditions would work, if so, what change in conditions would you recommend?
Thanks
Hi Eitan,
PEI is pH sensitive, if pH is increased the positive charges on amine group vanishes. So, in theory, adding strong bases would do the trick. The problem is that the amine groups behave as buffers, so it might take a very high concentration to release the DNA. DNA hydrolysis and depurination happen usually in acidic conditions, so using a base might not be a bad idea (just avoid heating the samples).
Perhaps adding ethanol might help, since PEI is soluble in ethanol, but DNA is not. But then you would have the problem of ressuspending the DNA again...
Another option would be increasing ionic strength: salt would act as counter-ion and shield the positive charges of amines. Or adding a negatively charged polymer, such as carboxymethylcellulose (CMC), that would compete with DNA for binding sites.
I have worked with PEI on a distant past, and not for DNA purification, so I don't have a protocol to give you. But I hope that some of these ideas help you.
Cheers,
Julio
Hi Eitan,
PEI is pH sensitive, if pH is increased the positive charges on amine group vanishes. So, in theory, adding strong bases would do the trick. The problem is that the amine groups behave as buffers, so it might take a very high concentration to release the DNA. DNA hydrolysis and depurination happen usually in acidic conditions, so using a base might not be a bad idea (just avoid heating the samples).
Perhaps adding ethanol might help, since PEI is soluble in ethanol, but DNA is not. But then you would have the problem of ressuspending the DNA again...
Another option would be increasing ionic strength: salt would act as counter-ion and shield the positive charges of amines. Or adding a negatively charged polymer, such as carboxymethylcellulose (CMC), that would compete with DNA for binding sites.
I have worked with PEI on a distant past, and not for DNA purification, so I don't have a protocol to give you. But I hope that some of these ideas help you.
Cheers,
Julio
I would agree with Julio,
try washing the PEI pellet with high salt buffers. As you sometimes need 1-2M LiCl to get rid of DNA from DNA binding proteins I would expect the same here.... maybe start with 1M NaCl and then see what happens.
Dear Eitan, did you ever follow this strategy up? I also am interested in a way of enriching DNA from a complex protein-DNA mixture, but which avoids Phenol/chloroform extraction. PEI seems like a good option, assuming it is possible to release free DNA from the DNA-PEI precipitates for downstream manipulation.
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