I'm amplifying a transgene using Pfu and plan to double-digest it with NotI and XhoI (same buffer) to ligate it into a plasmid. I've never done high-fidelity PCR and I'm not sure what the primers should be dissolved in (i.e., water, TE, or just Tris; pH=8?). I'm also uncertain if the cellular debris in the lysate should be removed before PCR (phenol:chloroform with 2-3 chloroform washes?).
I would always dissolve primers in TE as the tris buffers alkaline and the EDTA chelates divalent ions and inactivates nucleases. Water absorbs CO2 from the air and long term dupurination of the oligos may be a problem. Only dissolve in water if tris interferes with downstream applications..PCR has excess MG so is not much affected by small amounts of EDTA. Apart from colony pcr I would always remove cellular debris and as much protein as possible as these may include pcr inhibitors
Thank you so much, Paul! Your advice has always helped me.
Are 2 chloroform washes after phenol extraction sufficient to remove phenol from the aqueous phase? (I'm not sure if residual phenol would interfere with PCR; would it be appreciable with Pfu?)
Thank you Yazen Alnefeesi . Residual phenol is death to protein and enzyme reactions and must all be removed. The phenol will partition into the chloroform or CHCl3/isopropanol phase and I would expect that all of the 7% of the phenol that originally partitioned into the aqueous phase will be removed by using 2 extractions. I attach some information from Bitesize bio which has excellent information concerning the use of phenol and chloroform/organic solvents for your interest
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