Kunkel mutagenesis- Help needed
Yasha at bioch.tamu.edu
Sun Oct 9 11:07:21 EST 1994
In article <370vm4$p3g at mserv1.dl.ac.uk>, LEMAILLET
<lemaille at msdos.montpellier.inra.fr> wrote:
> Here come my questions:
> - Do you think that the titer of the phage is high enough ? If not, how could I
> increase it?
> - Do I have to screen more mutants ?
> - Is XL1blue suitable for this method ?
> - My primer is 18 base long with one mismatch in the middle, Is that enough?
> - Could I use this primer in another method, more efficient ?
> Sorry for this long explanation, but I really want to understand why it doesn't
> Any help greatly appreciated.Thanks in advance for your time.
To answer your first question, NO! You should see at least a 10^6
difference when titering for uridine incorporation. I would recommend
doing at least two phage amplifications in CJ236 with uridine present.
As to whether you should screen more plaques I would definitely say yes.
If you have only screened six, you will miss mutants if your mutagenesis
rate is less than 16%.
Your primer SHOULD be long enough, but there are a few things you should
check before running off to another method. First, have you checked to see
if your primer is annealing to the correct site? Using your primer for
sequencing can tell you both whether it is in fact annealing and whether it
is annealing to the correct site. If it isn't annealing you should check
to see if the primer is self-complementary. We use the Primer Reports
function in GeneWorks to check all of our primers before we get them
synthesized. If your primer is annealing to a second site, you can try
making a longer primer off set from the site you designed the original to
or you can try increasing the stringency of your annealing step.
Beware of moving on to other methods too soon. Each method for doing
mutagenesis has its advantages and disadvantages and I have learned the
hard way that the disadvantages of a given technique are rarely
acknowledged by the companies who sell the kit or even by the people who
use the various techniques. Also, if there is a fundamental problem with
the design of your oligo, you will only be transferring the problem to a
new technique. At least be sure that you have ruled out all possible
operator error before moving on to something new.
Texas A&M University
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