kkkk2011, on 25 June 2011 - 07:25 PM, said:
I tried to insert 1.4 kb DNA into pET vector. I used PCR to amplify the insert with NcoI and XhoI Restriction enzyme sites at the end( 6bp to NcoI site, and 3bp to XhoI site), cut the pcr products with these two enzymes for 20h at 37C. extract the digested dna with phenol then precipitate with NaAc and isopropanol, resuspended in 10mM Tris,pH 8.5 buffer. then do the ligation with pET vector, which also got digested with these two enzymes. I tried ratio from 3:1 to 8:1 in 10ul to 20ul ligation reaction, none of them is working. Please give me some help! Thanks!
hola, Why donīt you design your oligos to amplficate the fragment with the RE sites in the ends withouth need digest them?after purification of the gel you can do the ligation.Iīm not sure but I donīt see any problem. Buena suerte