Team:UEA-JIC Norwich/sdm

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University of East Anglia-JIC

UNIVERSITY OF EAST ANGLIA-JOHN INNES CENTRE

Site-directed mutagenesis

(Control reaction)

1. 2. 3. 4. 5. 6. 7. 8. 9.

5µl of 10× reaction buffer. 5µl of pWhitescript control plasmid. 1.25µl (25ng) of control primer 1. 1.25µl (25ng) of control primer 2. 1µl of dNTP mix. 1.5µl of Quick solution reagent. 34µl dH2O. 2µl DMSO. 1µl Quickchange lightning enzyme.

(Sample reaction)

1. 2. 3. 4. 5. 6. 7. 8. 9.

5µl of 10× reaction buffer. 5µl of template DNA. 1.25µl of Forward primer. 1.25µl of reverse primer. 1µl dNTP mix. 1.5µl of Quick solution reagent. 28µl dH2O. 2µl DMSO. 1µl Quickchange lightning enzyme.

Thermocycling parameters

Temperature Time(Secs) 95˚C 30 95˚C 30 55˚C 60 68˚C 240 4 ˚C

Time/seconds 30 30 60 240

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