We amplify genes by PCR using plasmid DNAs as templates. The downstream is to synthesize RNAs by in vitro transcription (IVT) using PCR products as IVT templates. The PCR products need to be purified by HPLC. We need to establish the HPLC purification. Suggestion regarding the composition of mobile phase related to DNA (1-3 Kb) HPLC and the device would be greatly appreciable.
Article HPLC purification of in vitro transcribed long RNA
I am surprised that you should need HPLC purification to prepare templates for IVT, unless you need quite large amounts of material. The spin-column gel and PCR purification kits (Qiagen or Macherey Nagel) yield high-purity DNA that is satisfactory for most purposes, including sequencing, restriction, ligation etc. If you need to get rid of the template, you could use DpnI digestion or gel purification. The Macherey Nagel minispin columns are given for a binding capacity of 25 µg each, so by using a few of them you can get several tens of µg of material, which is likely to come out 1) at a higher concentration than what you would get from an HPLC run and 2) in 5 mM Tris-HCl, ready for use in further experiments .
Thank Naser Jawad Kadhum , Arman Firoz and Arman Firoz . I have read those three papers. However, it is on purification of long RNAs. I need to purify DNA produced from PCR. The PCR products will act templates in IVT.
Dr. Pierre Béguin, we have no idea about whether PCR also produce incomplete DNA fragments. It will subsequently lead to truncated RNA in IVT if yes. That is why we consider HPLC of PCR products. Yes, we synthesize RNAs at a large scale (2-5 mg). Thank you very much!
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