My first post on protocol-online.
I've got a problem with Invitrogen's pENTR D TOPO cloning kit. My problem = I can't seem to get a 2kb PCR amplicon cloned into the kit's entry vector (this is supposed to occur during the 5 minute "TOPO Cloning Reaction").
A) I've got a PCR'ed up amplicon which I want to insert into the entry vector (verified size of amplicon on agarose gel electrophoresis)
B] Invitrogen D-TOPO kit advises a 5 minute 'TOPO Cloning Reaction' which I perform.
C) I then follow the instructions and transform the entry clone into chemically competent E. coli which are then plated onto kanamycin agar plates
D) Colonies are then chosen, cultured, and then the plasmid DNA is purified and sequenced.
The sequencing data reveals that the E. Coli were transformed with the D-TOPO plasmid not containing the PCR amplicon. This suggests that Step B (TOPO Cloning Reaction) has failed. I have done this twice already.
For the TOPO Cloning Reaction, the instructions advises use of 0.5uL to 4uL of PCR product containing 5-10ng of DNA (for 2kb product). I have so far used 10 and 20ng of DNA with no success. Should I attempt higher amounts?
Any advice would be greatly appreciated! Thanks
Edited by pepi, 24 October 2013 - 12:26 PM.