purification of single stranded cDNA? - (May/12/2008 )
Hi! What do you think is the best way to purify single stranded cDNA? I really want to get rid of the gene specific primer I used during the synthesis.
I have tried to make a second strand using Klenow and after that degraded ssDNA with ExoI, but that was not efficient enough.
I need a method that in which there will be no spill of my precious cDNA.
What is the goal? An ethanol precipitation will get rid of most of the primer. I'd use a glycogen or pellet paint co-precipitant so I could see the pellet.
Will the primer not precipitate because of the of the small size? What if I have a large primer, 44bp? Do you know the size limit for ethanol precipitation?
The goal is that I will make sure that the primer from cDNA synthesis will not be used in the later PCR.
It won't be perfect, but will get most of it. Which is probably enough, depending on what you are doing with the next PCR product. I doubt, for example, that you could see a band corresponding to that product on a gel. I don't know what the size cutoff is, and it certainly isn't sharp.