Site directed mutagenesis kit

Jean-Philippe Nougayrède jp.nougayrede at envt.fr
Mon Feb 15 10:20:20 EST 1999


Hi !
We used the Altered sites II and its protocol from Promega (revised
11/1995), with some modification.

1-We cloned our 2 DNA fragment (1.6 and 2.4 kb) into pALTER-1 vector
(HindIII/BglII fragment into HindIII/BamHI digested vector). The host
strain for the constructs was JM109.

2-Isolates (M9+thiamin+tetra 10 gamma)of JM109 hosting our constructs
(plus control strain: JM109 pALTER1) were cultivated o/n in LB+ tetra
(10 gamma). DNA  were then prepared using Quiagen columns (18 ul, in
H2O, not TE), and DNA were stored o/n at +4°C. I did not bother with DNA
quantification at any step :-)

3-The next day, 18 ul of DNA were denatured by adding 2 ul of fresh 2 M
NaOH- 2 mM EDTA, soft mix ("flicking"), and 10 min (not 5 min, see
below) at room temperature. 
The precipitation was achieved as recommended by Promega (fresh ammonium
acetate, gentle mixing with EtOH, 30 min at -70°C in a prechilled
isopropanol bath). The alkali-denatured DNA were dissolved in 100 ul TE
pH8, and placed at 0°C. At this step, you should run a minigel (10 ul of
DNA) to verify that the lower band is enhanced as compared to
non-denatured DNA. During preliminary attempts, I found that the
denaturation was not always achieved. NaOH reacts with CO2 of the air,
so only use a fresh stock! Also, ss DNA is more fragile than
supercoiled, so do not vortex.

4-The next hybridation reaction was done immediatly; 10 ul denatured
DNA, 1.25 pmol desalted-phosphorylated mutagenic oligos (from a
commercial source, stored at -70°C). The "Repair" and "Knockout" oligos
were from the kit (stored at -70°C). Temperatures were controlled with
our thermal cycler (5 min 75°C, then -1°C per min to 45°C, 45°C for 5
min) then room temperature for about 5 min, them in a 0°C water bath.

5-The synthesis of the mutated strands were achieved as described by
Promega, using DNA pol and ligase of the kit (stored at -20°C).

6- One ul of each reaction was transformed into ES1301 mutS strain by
electroporation (Biorad gene pulser) (50 ul E. coli, 0.2 cuvettes, 2.5
kV). 950 ul LB was added, and the bugs were let at 37°C (static culture)
for 30 min. Then, 4 ml of LB was added with 100 gamma ampi, and the
cultures were done o/n at 37°C with shaking.
Nota Bene; In my sense, the choice of ES1301 mutS is also a critical
point in this method. I used a fresh isolate of "medium" size to prepare
the "electro-competent" bacteria. Indeed, this strain gives a large
array of colony size on LB (phenotype associated with the deficient DNA
repair system).

7-DNA from 1.5 ml of the cultures were prepared using the miniprep
protocol proposed by Promega, modified as follow; lysozyme was added to
the first solution to enhance cell lysis, and I've done only a phenol
extraction step. DNA were precipitated with isopropanol and were
dissolved in 30 ul of water, and stored at +4°C o/n.

8-Five ul of those DNA were transformed into higly-competent MC1061 E.
coli (home-made freshly with the "boosted" RbCl2 method), selected onto
LB ampi.
Nota bene; the choice of MC1061 was not trivial, since this RecA+ strain
gives a good growth rate and is highly competent in our hands using the
RbCl2 method.

9-The next day, all the 49 colony were gridded onto LB Tetra (10 gamma)
/ LB carbenicillin (50 gamma) and the corresponding toothpicks were
cultured into LB broth carbenicillin (50 gamma). o/n 37°C.

My 2 controls pALTER1 without insert were carbR and tetraR (bad joke !),
but pALTER with insert A and B gave 12 and 5 colony respectively with
the good antibiotic pattern. The corresponding bugs were harvested and
DNA were prepared. The mutations were confirmed by restriction (our 2
mutagenic oligos were designed to introduce new BglII sites in the
inserts). 8/12 and 5/5 isolates were mutated as expected.
Since these mutations were done to achieve allelic exchange after
insertion of a kana cassette in the new BglII sites, we did not sequence
the mutants products.
The insert sequences and the mutagenic oligos are described in Mol
Microbiol, 1999 31:19-30.

Hope this helps.

JP Nougayrede.
Lab Microbio Molec.
Toulouse. France.

Holger Neumann wrote:
> 
> "Jean-Philippe Nougayrède" schrieb: 
> > We used "Altered sites II" from Promega, with one-shot success.

> I tried to use the 'Altered Sites II' from PROMEGA for a single-base
> mutation in a 1.6kB sequence but got no real success after several
> months of work.
> 
> It would be very kind if you, Jean-Philippe Nougayrède, if you could
> give me a few more details of your protocol (eg what plasmid was used
> etc).  Did I forgot anything important when performing or is there any
> improvement to the procedure I don't know (my protocol is from 05/96) ??



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