SS DNA and Mutagenesis (self-priming SOLUTIONS)

the End jgraham at bronze.ucs.indiana.edu
Fri Jun 4 10:57:23 EST 1993


Darren,

First, throw away the Bio-Rad kit (just kidding). Self priming of the 
control template is a relatively common problem and will vary from batch
to batch of single strand phagemid prep. Here are the steps you need to 
take:

1. When you separate you phagemid or phage from the superinfected bacterial
culture, spin it at maximum microfuge speed for ten minutes, remove as
much phage containing supernatant as you can without disturbing the pellet
at all, and then respin the supernatant again and remove the liquid. Better
to lose volume than disturb the pellet. It is essential that you remove 
all the cells now, as they contain plasmids that will contaminate your
single strand prep and lead to colonies in your control transformation. You
can see for yourself that the first supenatant is heavily contaminated 
with live cells by plating it. Keep RNAse the h*&( away from your templates
for the duration, contrary to the kit instructions, as it will generate
billions of priming short RNAs that will follow your templates once annealed.

2. When you precipitate your phage with PEG-salts, resuspend the phage
pellet in TE with a pipettor, and let it sit ten minutes on ice. Then spin
out any insoulubles. You will get a big pellet of junk here which contains
unknown priming material. This step is straight from the original Kunkel
Methods in Enzymology paper, and is omitted from "kits" that I have seen
pointing out the folly of not working from the original references.

3. If you still have self-priming, repeat the phage prep, don't overgrow the 
pahge (14 hrs only) leave out the antibiotics from the phage growth (omit
kanamycin in all cases) and don't release the single stranded templates
until the the same day that you are going to do the mutagenesis reaction. 
Templates left in storage (4 or -20) inevitably degraded in my hands even
following many penol extractions.

4. If all this fails you can still gel-purify your single stranded templates.
I have tried this unsuccessfully on alkaline agarose before I implemented
the above steps. I have heard that a standard agarose purification will
reduce backgrounds in the absence of the above precautions.

Good luck,

Jim
J. Graham
Richardson Biochemistry Laboratory
Indiana University Bloomington
.



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