Problems with pJET1,2Blunt
Posted 04 April 2011 - 09:17 AM
The first time I amplified my gen which have 512bp long using the DreamTaq Pol, and the electrophoresis gel showed a single 500bp aprox band, no primers appeared nor non-specific bands presented
I used the CloneJet PCR kit from Fermentas which uses the pJET1,2blunt cloning vector, since my PCR product was amplified with Dream Taq I used the blunting enzyme to eliminate the TA overhangs left by the polymerase.
I incubated the ligation reaction, transformed in XL1-Blue Ecoli strain, did minipreps and digested the recovered plasmids
The gene Im looking for is flanked by the NcoI and PmeI restriction enzymes, so I digested it and found that the NcoI was present but the PmeI site was not
I repeated the experiment using blunt PCR productswith the same kit and still no sign of the PmeI site
After that I tried cloning my PCR product in the pGEM-T vector and the NcoI and PmeI sites appeared,
Anyone knows what am i doing wrong?
Posted 04 April 2011 - 10:06 AM
You do not need to sequence the whole plasmid, just use the primers provided (pJet1.2 forward and reverse sequencing primer).
Edited by adrian kohsf, 04 April 2011 - 10:12 AM.
..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434