| SITE-DIRECTED
MUTAGENESIS
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SITE-DIRECTED MUTAGENESIS
D. Nickloff (92) Anal. Biochem. 200:81-88
- Kinase oligos with PNK, [ ]f ~0.25ug/ ul
- Denature and anneal primers to template : 1X annealing buffer, 150ng template ( pmol), ~0.5ug (50pmol) @primer /20 ul final.
- 100oC -3'; then ice/5'. Spin down 10"
- To each tube add: 5 ul H2O, 3 ul 10X synthesis buffer, 0.5 ul T4 DNA Pol (~ 5unites) and 0.3ul T4 dna ligase (NEB) --37oC/3H
- 10 ml into 100ul muts E. coli such as BMH 71-18
- Grown liquid library & digest w/ selection enzyme (Helps to include an internal control to ensure full digestion) --> retransform to DH5alpha or TOP10
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