Best Way Clone PCR Products

Paul N Hengen pnh at fcsparc6.ncifcrf.gov
Tue Oct 4 17:38:34 EST 1994


 In article <aquilla.1131006611C at sadye.emba.uvm.edu>
 aquilla at salus.med.uvm.edu (Tracy Aquilla) writes:

| ... I did find that genecleaning the DNA before restriction digest
| did not work for me. There was something in the genecleaning process
| (probably the EtOH leftover from the wash) which inhibited digestion. I
| found this was a common problem when I asked around. I switched to
| beta-agarase purification from the gel and have never had a problem
| cutting since then.

> ....I always spin after removing the supernatant, then use a thin capillary
> tip to remove the last tiny droplet of supernatant after washing.

When I use the Gene-Clean binding matrix, or even if I use my own homemade
version, I dry my pelleted stuff in a rotovac for 5 min. Then, resuspend it
in dH2O and float the tube in 45-50 C water bath for a few minutes, flick the
tube, centrifuge a few seconds, and move on. I've not encountered any problems
with residual ethanol inhibiting enzyme reactions.  BTW, I'll never use
beta-agarase again! What a huge waste of time... viva la syringe ;-)

@article{Hengen1994Septibs,
author = "P. N. Hengen",
title = "Methods and Reagents - Recovering {DNA} from agarose gels",
journal = "Trends in Biochemical Sciences",
volume = "19",
number = "9",
pages = "388-389",
month = "September",
year = "1994"}

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