fatima at natur.cuni.cz
Thu Aug 9 14:14:18 EST 2001
A rather ancient protocol, developed in Kim Nasmyth's lab in Vienna,
mainly by Angelika Amon and myself, follows (see also Cvrckova et al.,
Genes Dev. 1995, 9:1817-1830).
Fatima Cvrckova (fatima at natur.cuni.cz)
Buffers: 10x PCR buffer: 500mM KCl
100mM TRIS pH8
REACTION MIX (EXAMPLE: LIMITING G):
linear DNA: 20ng
primers: 2x 400ng
dNTPs (i.e. A, T, C) (10mM) 5 ml
dGTP (10mM) 1 ml
10x PCR buffer 10 ml
MnCl2 (5mM in 10 mM TRIS pH8) 10 ml
b-Mercaptoethanol (1M) 1 ml
Taq-polymerase 8 units
add water to a final volume of 100 ml
Note: 1. Use Pharmacia nucleotides (A.A.). Boehringer works fine (F.C.).
2. Prepare MnCl2 and b-ME always freshly. You can keep a
concentrated solution (100mM in H20 !!! - it would go off in Tris!!!) of
MnCl2 for a few days at -20 ºC.
3. I always do four reactions in which one of the four
nucleotides is limiting .
4. Use native Taq, NOT AmpliTaq. Perkin-Elmer native Taq is
Choose appropriate annealing temperature and synthesis time (for a 2 kb
fragment I do 2 minutes - A.A.; for 3.2 kb fragment I needed 5 minutes -
F.C.). I usually do 20 cycles.
Mutation rate: approx. 1 per 300 base pairs (A.A.), 1/450 bp (F.C.)
NOTE: if mutagenizing a fragment cloned in pUC or YXplac, use Gotthold's
add 16 µl 15 mM MgCl2 per 80 µl reaction; annealing 50 - 52 ºC,
elongation 72 ºC.
i.e. SCALED-DOWN REACTION SETUP (F.C.):
10x PCR buffer 16 µl
10x primers (i.e. 40 ng/µl each) 16 µl
10x template (i.e. 50x diluted miniprep plasmid) 16 µl
15 mM MgCl2 16 µl
10x ME (i.e. 140x diluted mercaptoethanol from the bottle, FRESH) 16 µl
10x MnCl2 (i.e. 100 mM stock 20x diluted in 10 mM Tris pH 8) 16 µl
Perkin-Elmer native Taq pol. 20 u = 4 µl
mix well and divide into 4 tubes 24 µl each, then add to each tube:
non-limiting dNTPs (5 mM) 4 µl each
limiting dNTP (1 mM) 4 µl
mix by pipetting up and down and run the PCR.
Afterwards process like any other PCR to be cloned.
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