My experiment is to make dsDNA from a pool of variable size fragments (~200 bp to 25 kb). I am using degenerate decamers for second strand synthesis with Phi 29 polymerase. Next step is to purify the dsDNA smear for which I am using ZYMO oligo clean and concentrator kit so that an ssDNA below 16 nt is discarded. Next step is to ligate the dsDNA to a 25 bp linker and purify the DNA out of un-ligated linkers. My questions are as follows:
1. Is Phi 29 the best polymerase for this purpose? I have tried vent exo- and klenow exo- but got best amplification (smearing on an agarose gel) with Phi 289.
2. I could not find a good method to get rid of the unligated linker. I am using ZYMO DNA clean and concentrator kit. It gets rid of unligated linker but there is a huge loss of DNA, only ~30% recovery. Can anybody suggest any other kit or method? I have already tried running the smear DNA on gel and purifying the DNA using silica beads but again the recovery was extremely poor.
Are you using a spin-column method for gel extraction? I've found that I get much better yields by doing a beta-agarase digestion of agarose gel followed by EtOH precipitation (+glycogen). I don't know for sure if this would be an improvement over the ZYMO kit, but I routinely do the beta agarase prep and am able to effectively remove adapter contamination while recovering ~50% of my DNA.
Hi Govinda, I will look in to the method of beta-agarose digestion. Thanks.
Hi Mark, I hd no idea that Chelex can be used for DNA purification too. I will read more about it now. Thanks.
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