You will need:
10 x Taq buffer (Promega)
Taq Polymerase (Promega)
1) Digest plasmid to be used with a blunt-end restriction enzyme (I use EcoRV and pBluescript SK-).
2) Clean digestion reaction by phenol/chloroform extraction and ethanol precipitation.
3) Redissolve DNA in 1 x Taq PCR buffer (I use Taq and buffer from Promega but I think any non-proofreading Taq will work) to give approx. 1ug DNA/20 ul.
4) Add dTTP to a final conc. of 2mM (typically from a 100mM stock solution).
5) Add Taq polymerase to give 1 unit/ug DNA/20ul.
6) Incubate mixture at 72°C for 2 hours.
7) Extract mixture once with phenol/chloroform and once with chloroform.
8) Ethanol precipitate DNA, wash with 70% ethanol, dry and redissolve in TE at an approx. conc. of 25ng/ul.
9) Quantitate DNA (I use an approx. quantitation by gel fluorescence against standards).
10) For ligations, use approx. 25-50ng T-vector and sufficient PCR product to give a 3:1, insert:vector molar ratio (I use T4 Ligase from Gibco-BRL together with their PEG-based buffer).
11) Ligate O/N at 15°C.
12) Transform 1/5 ligation mix into competent E.coli (I use DH5a cells at approx. 5 x 107 cfu/ug pUC19).
Dr. Simon Dawson,
Department of Biochemistry,
University of Nottingham,
The Medical School ,
Clifton Boulevard ,
Tel: +44 (0)115 9249924 ex.44787,
FAX: +44 (0)115 9422225,
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