Construction of homemade T-vectors

 

Construction of homemade 'T-vectors'

This method is after Marchuk, D., et al., 1991, Nucl. Acids Res. 19(5), pp 1154.

You will need:

10 x Taq buffer (Promega)
Taq Polymerase (Promega)
Phenol/chloroform mix
100mM dTTP
TE buffer
Absolute ethanol
70% ethanol

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).