Dear all, Is there anyone have experience do ethanol precipitation PCR product prior to phosphorylation by T4 polynucleotide kinase? According literature ethanol precipitation by ammonium asetat prior prior to phosphorylation by T4 polynucleotide kinase can inhibit the T4 PNK. How about natrium asetat, glycogen?
Hi there,
Ammonium ion is indeed inhibitory for kinase activity so avoid it upstream the PNK treatment of your DNA. Both glycogen and NaAc are OK, the only drawback is that glycogen is from natural source so it's not 100% pure (usually contaminated with nucleic acid) and that NaAc tends to coprecipitate proteins along with DNA (but there is not much of it in a PCR reaction).
Dewi, depending on the amount of product from PCR (DNA), you may have considerably less than incomplete recovery when you precipitate DNA at relatively concentration. Adding "molecular biology" grade glycogen will be quite useful and there is very little chance that you will appreciable nucleic acid contamination in glycogen.
A better and faster alternative may be to use mini-columns to "clean up" PCR products. These are sold in kits by many companies, such as Qiagen, and work rather well.
- Badges
- Science method