I'm trying to clone a 8 kb viral cDNA fragment into a 3 kb vector. I tried it now several times and I'm really desperate because it doesn't work.
I get the fragment by pcr and through the pcr there are 2 restriction sites on either end of the fragment created. The restriction sites are BsiWI and NotI. There are 5 bp after each site to have a better cleavage.
After pcr (which is quite successful) I check it on a gel to see if it was successful. Then I do pcr purification with the qiaquick Pcr purification kit.
I check the amount again on a gel and estimate how much to use for restriction. I cut about 1 µg. First with NotI (over night) then the next day in the same buffer (is the same for both enzymes, NEB) with BsiWI. I do the same with the palsmid. I dephosphorylate the plasmid with Antarctic Phophatase (NEB, very new product but it works very, very good) and then do a gel extraction. The cut fragment I clean with a Qiaquick Spin cloumn. Then I check vector-Insert relation on gel. I then ligate over night , 16°C.
Edited by arwen76, 11 January 2005 - 01:43 AM.