Dear helpful community,
I am having trouble PCR-amplifying and cloning a ~2.5kb fragment into the pET101 directional cloning vector by Invitrogen.
-Q5 high-fidelity polymerase (produces blunt ends)
-Gene-specific forward and reverse primers Tm ~60C
1. 98C initial melting temp, 2 minutes
2. 98C melting temp, 40 seconds
3. 55C annealing temp, 30 seconds
4. 72C, 3 minutes (loop to step 2 34 times)
5. 72C, 5 minutes (final extension)
6. 4C, hold
After cycling, I have very strong products that appear to be about 2.5kb in size, with only faint secondary bands that I presume are the original plasmid in different conformations Picture 1.pdf 342.95KB 442 downloads.
I then excise my bands form the gel, combine excised fragments, and purify using a basic kit and end up with about 50ng/ul DNA of fairly high purity.
I then ligate the purified fragment into Champion pET101 Topo directional cloning vector according to the protocol by Invitrogen, and finally transform. The next day, I see about 30-50 colonies on each plate
The problem(s): 1) Running colony PCR with T7 forward and reverse primers shows that most clones are missing the 2.5kb insert, and 2) those that appear to have an insert are the wrong size (about 1.5kb) ( Picture 2.pdf 271.01KB 463 downloads, circled in red), AND sequencing reveals that plasmids from those colonies circled in red contain a fragment of Topo vector that should not be in there.
Running PCR on the purified plasmids from those colonies using T7 primers reveals similar results in that they contain a small mysterious fragment Picture 3.pdf 555.69KB 450 downloads
I would truly appreciate any light you can shed on this matter. Thank you, and cheers!
Edited by aodonnell311, 24 March 2016 - 07:20 AM.