pGT4ΔB

Overview
Digestions
pGT4ΔB
Electrophoresis
DNA clean-up
Ligation
Fragment isolation
Competent cells
Transformation
Recombinants
pGTλ3758ΔH
Miniprepping
Blotting
Probe Labeling
Hybridisation
Probe Detection
PCR

Construction of pGT4ΔB

pGT4ΔB is a plasmid vector pGT4 mutant. The mutation is a disrupted BamH I restriction site. So, ΔB stands in this case for: "without BamH I site".

The construction of pGT4ΔB starts with the digestion of pGT4 with restriction enzyme BamHI.
Klenow DNA polymerase is used in the next step...

The experimental steps in the pGT4ΔB construction can be done simultaneously with those of the library construction
(use your mouse to hover over the scheme):

 

 

Klenow enzyme

To create blunt ends on BamH I DNA fragments the large fragment of E.coli DNA polymerase is used here. This so-called Klenow enzyme is a truncated version of polymerase I: it lacks the 5 to 3 exonuclease activity. It can synthesize DNA complementary to a DNA template in a 5 to 3 direction, provided it is given a primer with a free 3-OH, and dNTP's.
Klenow enzyme is also used in the probe labeling reaction.

 

  See the Movie about
Blunting DNA ends using Klenow enzyme

Read the movie text