QuikChange II Site-Directed Mutagenesis

Hello. I am trying to do a site directed mutagenesis with three
different primer pairs on one plasmid with the Kit mentioned above.
Unfortunately it was just possible to get a positiv result by annealing
one primer pair at one time. We tried to repeat the reaction with the
other two primer pairs two times but didn´t get colonies after
tranformation so far. Could please someone give us hints how to change
the reaction condition to prime the other two primer pairs or how to
check for mutagenesis success.

Thanks a lot

John

Having used the kit many times I would increase the cycle number from
18 to 25. This normally works for me.

Good Luck

Mike

In article <1129726995.067502.239020@g43g2000cwa.googlegroups .com>, "[Only registered users see links. ].uk" <[Only registered users see links. ].uk> wrote:

Could you clarify your statement please?
Which of the following is correct?

1. You usually do 25 cycles and it normally works for you.
2. Whenever typical (e.g. recommended) PCR protocol does not
produce colonies/mutants, you switch to 25 cycles and it
normally helps.

Thank you.

Dima

John wrote:

Try lowering the annealing temperature. I always set the annealing
temperature a couple of degrees lower than I should.

In article <[Only registered users see links. ]>, ChenHA <[Only registered users see links. ]> wrote:

I agree. We also noticed that our success rate is better when we go
down from what was originally recommended by Stratagene.
Actually, now that I realize that QuikChange II is simply good
old QuikChange with PfuUltra (which means I've been using version
II before Stratagene started to make it!), I do have what I'd consider
the most useful trick in difficult cases:

Do few cycles (1-3) with just one of the primers, then mix the two
reactions and continue as normal. Works wonders for difficult cases
such as inserting 15 aa (the longest I've tried).

Oh, and if anyone is interested: QuikSolution™ Reagent is
unadulterated pure DMSO. In other words, buying PfuUltra
from Stratagen, DpnI fron NEN and having decent home made
electro-competent cells is equivalent or better to the useful
kit content in QuikChange II.

Speaking of QuikChange:

I noted Stratagene now sells QuikChange® Multi Site-Directed Kit.
Judging from description, it contains a mix of polymerase and ligase.
The idea is, basically, to synthesize enough single stranded circular
DNA and let E.coli take care of 2nd strand synthesis. This is
accomplished by using a mix of polymerase and ligase. (I think
the idea was triggered by observations that i) just one primer works
sorta OK in otherwise normal protocol, ii) using two pairs of primers
to make two mutations simultaneously works too, albeit with
lower efficiency, ~ 30% in our lab).
Of course, Stratagene does not really tell what ligase they add
and what substrate it takes - which allows them to raise the price
of the kit by 1.5X. Anyone knows of any commercially available
thermostable DNA ligase?

DK

DK wrote:

Good idea, I'll try that with something that I'm having problem with
at the moment (the oligos were designed wrongly so may not anneal
properly).




What transformation efficiency do you get with your electro-competent
cells? I inherited an electroporator from a previous occupant of the
lab, and so far I haven't been able to do better than my chemical
competent cells (~1-3 x 10^8 cfu, using Inoue method). It may be the
electroporator (it is a bit flakey), but wondered why I can't get to
10^9-10^10 cfu which is what is claimed possible.



Don't know, but you can probably clone it out yourself if you are
going to use quite a lot of it. I think I may have a little
chromosomal DNA from Methanococcus jannaschii left over from some
years ago, so might take a look one day.

[Only registered users see links. ][SWALL-acc:Q57635]+-vn+2

In article <[Only registered users see links. ]>, ChenHA <[Only registered users see links. ]> wrote:

That's interesting because I was never able to get better than about
2x10^7 with Inoue :-). Just how crucial is temperature there? I used
to wrap flasks in wet towels in order to drop temp down from 22C...
Do you mind posting *your* Inoue protocol (if it's any different
from published)?


I have not made them myself in ages - I just don't need
super efficiency in any application. With DH5alpha, I used
to get 5x10^8-10^9 routinely. 10^10 I simply don't believe
and have never seen - even when testing cells that claimed it.

Basically, there are several things to keep in mind:
1. Very clean glassware! Usually a non-autoclaved water
works better.
2. Determine optimal collection density (remember that the
OD600 is the most meaningless number because it is
spec-dependent!). For a given spec, I used to collect few cultures
at OD600 0.4 - 1.0, wash quickly, resuspend at ~ 50x and
test. Absolute efficiency is not important here - just pick the
density that gives you most colonies.
3. Have as concentrated cell suspension as possible
(yet practical and convenient - YMMV).
4. Don't use more than 10% glycerol. I've seen that 20% glycerol
that some people use is strongly inhibitory. Don't autoclave
glycerol.
5. Determine optimal electroporation parameters. For plasmids
less than 10 kb, 1 mm cuvettes always work better. It also
varies batch to batch. For TOP10 cell in our lab currently, the
optimum has always been within 1.4 to 1.6 kV/cm with efficiencies
dropping significantly outside this range in both directions.
6. Quickness of execution. Cells are almost literally getting fried
there - so it's important to add culture medium as soon as possible.
OTOH, I've also seen people holding cuvettes *after* electroporation
on ice for 5-20 minutes and then complaining that electroporation does
not work good well enough - in this case, they were just killing cells
by not letting pores seal on ice!


Nah, not worth it in my case. But I found one:
[Only registered users see links. ]

It would be trivial to test Stratagene's buffer for NAD presence and it it
indeed has NAD but not ATP then I guess it will be safe to mix your own
kit-free "QuikChange Multi Site" reaction. This Ampligase is surprisingly
not terribly expensive!

Dima

DK wrote:

I don't think it's really different from the published one, not that I
remember what the original protocol is. So roughly -

Inoculate cells in 50 ml of SOB, grow at 18 deg O/N. You can first
grow at 37 deg a couple of hours first if you find it growing too
slow. Spin down cells at OD 3-6 (I have spun down cells at OD 7 it
still worked fine), and resuspend in ~0.3 vol filtered-sterilised TB
(10mM PIPES or HEPES, 15mM CaCl2, 250mM KCl, pH to 6.7 with KOH, then
add MnCl2 (55mM)). Incubate for 10 min. Spin down, resuspend in <8 ml
vol TB and incubate for 10 min.

I think you need a reasonable concentration of cells, so depending on
what OD you spin your cells down, you end up with 12-20 X
concentration of cells at the end. I add to 7-8% DMSO before freezing
cells in liquid N2. Treat cells gently when resuspending and
pippetting (don't pipette up and down). DH5a works fine, XL1Blue
seems to be much more fragile and grows a little slower. Use 100 ul
or more for each transformation. Try more MgSO4 in SOB (up to 2X I
think) if it doesn't work well in normal SOB.

The only other things I would mention is that, as you noted, different
spectrometers can give different readings, so you determine the
optimal OD empirically. I would spin cells down at a lower speed
(never more than 2000g for the first spin, and 1000g or lower after
that). I always use plastic baffled flasks, but don't really know if
it is better than using glass ones (I read somewhere that using
plastic is better), and I also never wash my flasks for making
competent cells with detergents or disinfectants. It could be all
superstition, but it has worked for me and I stuck with those
practices.

I recently had some problems getting good competent cells, not really
sure why, could be the water getting dirty, or someone has been using
my flasks and cleaning them with disinfectants (you know how things
are like in lab, things left around and they always get taken). But
seems to be back to normal after a few bad ones. So there appears to
be unquantified variables, if it doesn't work for you, it may be
difficult to work out why.

I need to have higher transformation efficiency because I may have to
create a library of combinatorial peptides, so 10^9, or better still
more than 10^10 would be ideal.

I'll try your protocol later.

On Sun, 23 Oct 2005 03:33:53 GMT, [Only registered users see links. ] (DK)
wrote:


Forgot to answer this - the temperature is important. Long time since
I last read the Inoue paper so I may have misremembered, but I think
the low temperature interferes with the cell wall synthesis and it
becomes weaker. Perhaps you can try it at higher temperature with
XL1Blue, it seems to grow quite slow at 18, so may be it will be
happier at a higher temperature. I don't use XL1Blue normally so I
can't say much about it.

You can also try one of Hanahan methods if you don't want to grow at a
low temperature (the one with cobalt hexamine and supposedly very
good). But I have never tried it, and I'm afraid I don't have the
reference.

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