Creating empty vector by self-ligation - (Apr/06/2009 )
ygh on Jun 4 2009, 04:58 PM said:
Could you please share with us how you solved your problem?
hmm..basically..i did a double digestion on the pCMVSport-6-insert using the SalI and xhoI( these create compatible ends which can be ligated later). Then i ran it on 1% agarose gel and cut out the 4.4kb band (which is the pCMVsport-6 empty vector backbone). Followed by gel extraction and the purified plasmid DNA was then religated using T4 ligase, incubate for overnight at 16 degree celcius. Then i transformed 10ul of the ligation mixture into E coli DH5alpha. I managed to get colonies on LB+Amp plate.
That's all of it.
Best of luck.
There is a huge difference, the supercoiled form runs lower. Initially i used 6ug of plasmid DNA for double digestion and i assume i lost some during the gel extraction process.