This is a cached page for the URL ( To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
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).

Keywords: DNA, PCR, cloning, T-vector

Contact Email Address:

Submitted at 16:39 on 22/1/96 by:

Dr. Simon Dawson,
Department of Biochemistry,
University of Nottingham,
The Medical School ,
Q.M.C. ,
Clifton Boulevard ,
NG7 2UH,
Tel: +44 (0)115 9249924 ex.44787,
FAX: +44 (0)115 9422225,

If you think you have a useful addition that could be made to this method, please submit it by clicking this button and filling in the relevant details.

Click here to return to the Submission Form or here to go to the Methods Finder.