very low 230/260 readings - (Apr/16/2006 )
Hi all, I am trying to subclone insert into vector via Not1 and HindIII sites.
I double digested them in buffer 2 (according to NEB chart) and gel purify (Qiagen).
After that I used Nanodrop to measure the concentration.
i get conc of ~ 14ng/ul, but the baffling thing was that 280/260= 2.5 and 230/260=0.02!! (for both vector and insert)
that is suppose to mean contamination but how? I have repeated the digestion and purification twice but its still the same problem.
anybody has any clue whats the problem? (i went on with the ligation step the first time and i had colonies that gave me unexpected results when i did restriction analysis...)
(I've used the same Qiagen gel purification kit b4 and it was alright)
14 ng/ul is at the lower limit of reliable detection on the nanodrop. I certainly would not take the readings of ratios as anything to worry about after gel elution. Elute in lower volume to increase concentration, or, more pragmatically, just go ahead and ligate your insert. Is your insert the correct size? You can check colonies by colony pcr rather than RE digests, which tends to be easier and quicker for obtaining correct length insert clones.
will go ahead with ligation then. THanks!