PCR cloning problem - could not get colony (Jun/25/2011 )
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!
kkkk2011 on Sun Jun 26 03:25:17 2011 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
Protolder: I have no idea what you mean. PCR will leave poor ends for ligation. Taq will typically add an extra A, and unless you do something, the 5' phosphate needed for ligation is missing in oligos. You might be able to make this work for Pfu or Phusion pcr with phosphorylated primers, but in general this would be a bad strategy.
phage434 on Mon Jun 27 12:20:40 2011 said:
Protolder: I have no idea what you mean. PCR will leave poor ends for ligation. Taq will typically add an extra A, and unless you do something, the 5' phosphate needed for ligation is missing in oligos. You might be able to make this work for Pfu or Phusion pcr with phosphorylated primers, but in general this would be a bad strategy.
Thanks a lot, the fact that I be an enthusiastic forer doesnīt mean that I be an expert . I thought that this way runs or probably it needs an intermediate step with a type pGEM-T vector. My excuses to boht of you and my gratitude. Have a nice day
yeah i think phage434 is right, but thank for u proteolder for at least share mind...maybe the problem is started from digestion...please sometimes check the reagent too...my friend got bad result for digestion but then, when we tried with another tube (new) it's working..but maybe it not happen to u..well must be wrong with digestion, or ligation could be in transformation too...so having control will help to detect the mis-step..hope u can get it soon...keep trying!