Stephen Bell wrote:-
>Netters,
>> I am interested if anyone has a protocol for PCR
>mutagenesis of a gene fragment then transforming yeast with
>the mutagenized PCR product and a gapped plasmid directly
>(rather than making a mini-library of mutagenized genes
>before yeast transformation). In particular I would like to
>know how much homology is needed between the gapped plasmid
>and the PCRed fragment to get good efficiency of
>recombinants and whether there are problems with religation
>of the plasmid without recombination.
>>Thanks,
>>Steve Bell
We have used the protocol below (based on Muhlrad, D., Hunter, R. and Parker, R.
(1992) A Rapid Method for Localized Mutagenesis of Yeast Genes. Yeast 8, 79-82.)
to make Ts- alleles of several essential yeast genes. Regarding the overlap
necessary beteen the gapped plasmid and PCR ragment we have not rigorously tested
the minimum requirements, but in one case (GLC7) the overlaps were 46 and 374 bp
while in another instance (PPH22) they were 148 and 187. In the latter case the
primers were flanking polylinker sequence. This gave hundreds to thousands of
transformants in each case and greatly stimulated the transformation frequency
over that obtained just with the gapped vector. However, since there was a
background level of transformation without PCR fragment, either ligation or repair
(in our case from a second plasmid in our strain carrying the wild-type gene in
question) clearly could be a problem depending on exactly what you are doing.
Gapped plasmid repair with mutated/non mutated PCR product
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A. Error-prone PCR for random mutagenesis
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Mutagenic and non-mutagenic reactions are set up in parallel. Standard mutagenic
conditions:-
i) Ratio of (dTTP/dCTP) : (dATP/dGTP) = 5 : 1 (could also try 1 : 5)
ii) Include 0.1-0.5 mM manganese chloride in the reaction
iii) 2X normal (final) conc. of Taq polymerase in reaction (i.e. 0. 05 U/mu-l).
For each set of template/primers, optimise the following conditions:
a) Ratio of MgCl2:MnCl2 in the reaction e.g. for GLC7, used 0.5 mM but for PPH22
used 0.3 mM. This can greatly affect the frequency of mutations obtained.
b) The PCR conditions for denaturation/amplification/annealing
1. Stock solutions
50 X stock "mutagenic" dNTP mix (low A+G): 25mM dTTP, dCTP, 5mM dATP, dGTP
50 X stock "normal" dNTP mix: 25mM each dNTP
2. Reaction for 100 ul:
[Stock] [Final] Vol.(mu-l)
------- ------- --------
Primer 1 200 ng/mu-l 4 ng/mu-l 2.0
Primer 2 200 ng/mu-l 4 ng/mu-l 2.0
template (1) 10 ng/mu-l 0.1-0.2 ng/mu-l 1.0-2.0
Reaction buffer (2) 10X 1X 10.0
MgCl2 (2) 25 mM 2 mM 8.0
dNTP mix (3) see above see above 2.0
water - - 68-72
[MnCl2 10 mM 0.3-0.5 mM 3.0-5.0] (5)
Taq pol.(4) 5 U/mu-l 0.05 U/mu-l 1.0
Footnotes (1) template = circular plasmid
(2) Some buffers may already have the MgCl2
(3) diluted from Pharmacia 100 mM stocks
(4) We have used Promega or Boehringer Mannheim enzyme successfully
(5) mutagenic reaction only
3. Mix, heat at 100 deg. C for 5 min, add the Taq polymerase, mix, add oil (100
mu-l), pulse spin briefly in a microfuge and put in PCR machine using appropriate
settings variable because dependent on template/primers etc. and ideally
pre-optimised) eg:
94 deg. C 45-60 sec ramp 0 (1 deg.C/sec: on a Techne PHC-3)
55 deg. C 2.0-2.5 min ramp 30 (0.5 deg.C/min)
72 deg. C 2.5-5.0 min ramp 0
After 30 cycles, link to 94 deg.C 1.0 min (ramp 0), 4 deg.C 15 h
4. Check for purity on gel after ether extraction of oil and if a clean band of
the expected size is obtained then it can be used directly in gapped plasmid
method. Alternatively, gel purify the band e.g. with GeneClean II.
5. In our system the 5' primer was around the ATG of GLC7 and the 3' primer 130
bp downstream of the stop codon. Convenient sites were chosen so that the
homologous overlap at the 5' end was 50 bp (better to be longer) and at the 3' end
300bp.
B. Gapped plasmid Transformation
--------------------------------
For making Ts- mutations, the host strain is a suitable yeast carrying a wild-type
copy of the gene in question on a URA3-CEN plasmid and a gene knockout in the
chromosome. The gapped plasmid was prepared by digestion and gel purification
using Geneclean II and carried a copy of the gene on a CEN plasmid with a
different market e.g.TRP1.
1. Usually added following per aliquot of yeast cells made competent TE-LiAC
method:
10 mu-l PCR product mutated or non-mutated (didn't quantify but 10 ul is a good
intensity band on a gel)
50-100 ng gapped plasmid
50 mu-g sheared single-stranded salmon sperm DNA
50 mu-l competent yeast cells
2. Mix briefly, then added 300 ul 40% PEG/TE/LiAc. Shake at 26 deg. C 30-45 min
and heat shock 42C 15 min.
3. Spin 3000 rpm for 2 min and resuspend in 100 mu-l TE.
4. Plate out on selective plates. Include along side gapped plasmid alone and a
zero DNA sample as controls. In our system we then patched out approx. 500
transformants onto selective plates containing 5-FOA and then when these had grown
up, repatch onto selective plates at 26 deg. C and 37 deg. C. At this stage we
got around 10% Ts- mutants but this will no doubt vary from gene to gene.
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Dr. Michael J. R. Stark,
Department of Biochemistry,
The University,
Dundee
DD1 4HN
Scotland
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+44 1382 344250 Phone
+44 1382 322558 FAX
m.j.r.stark at dundee.ac.ukhttp://www.dundee.ac.uk/biochemistry
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