Hi,
I know, this is a very basic question but it just comes up again and again... Which method do you actually use for cleaning-up PCR products? I always have the impression that when I use the everyday Qiagen PCR purification kit I lose a lot of sample, well, I know that you lose some, but has anyone ever tried to estimate the loss more or less?
I guess another option would be to use the Sephadex plates, I guess in this case you don't lose that much, but I have also read that the reaction purified on Sepahdex do not come out totally clean of primers and dNTP's, therefore the concentration you measure is overestimated. Does anyone have experience with this as well?
And there is the phenol:chloroform extraction, for sure. This is actually my favorite method, but it takes too long:(
I have looked around to find a similar topic, but if I overlooked an already existing discussion, please let me know.
Cheers,
basak
I suggest the Promega's one...it's less difficult than the Qiagen's one. By the way, Basak is right, the Phe-Chlo purification is the best one....especially if you have to clone it in a downstream work.
Now, i'm gonna try a CTAB protocol....let's see if it'll work well.
I have been using the MultiScreen PCR mu 96 plates from Millipore, and they work very well for basic 96-wells format cleanup for sequencing. For cloning I would still recommend Qiaquick, but I agree that the gel-purification version of this kit usually results in loss of about 50% of your fragment.
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