PCR product purification - (Dec/06/2005 )
hi everybody! I am trying to purify PCR products for using as templates in a new PCR, but something seems to be inhibiting the new reactions. I've already tried the wizard PCR preps DNA purification system (Promega) and the Nucleotrap nucleic acid purification kits (Clontech). The samples are eluted in water.
Could you give me any solution?
The usual problem is far too much template. Dilute the first reaction 100-1000x for the second. Purification should not be necessary if you dilute sufficiently.
You can also just take 1 µl of a 1/10-1/1000 of your original PCR and perform your second round.
(Also, if you do not have much PCR-product from your first PCR, you can go up to 5 µl of your first reaction and just put it into your second, but then you better adjust your Mg-concentration).
I have the same problems. But I change to use DNA purification kit from Fermentas. It's work!!
because this kit provide the beat granules, you can elute your DNA by dbH2O (No salt contaminate in DNA solution)