- design a primer1 with the mutated or additional nucleotides
in the middle flanked by 15 to 18 nt on both sides. Primer 2 is the
reverse complement of primer 1
- set up following three PCR reaction with increasing amounts
of template DNA (each 50 µl)
template plasmid (1/10/50 ng) primer 1 (0,5
µM final)
primer 2 (0,5 µM final)
dNTP mix (each 0,2 µM final)
Pfu DNA polymerase (2,5 U)
- run a programme with 15-20 cycles, 1min per kb at
68ºC, annelling temperature 55-60°C
- perform a buffer exchange with spin columns (Sephacryl 200)
or add 10 µg tRNA, extract with phenol/chloroform and precipitate
with ethanol, dissolve in 50 µl water
- digest with DpnI (1-4 h, 10 U). DpnI endonuclease is
specific for methylated
DNA (template plasmid), but does not digest the DNA synthesised during
the PCR.
- extract with phenol/chloroform, precipitate with ethanol,
add tRNA if necessary
- transform E. coli by electroporation, plate 100%
- checks clones by minipreps and sequence 10 clones
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