Problem with PCR - (Aug/09/2005 )
Hi , I`m first time on this forum
I've been doing PCR cloning with a gene for about three months.
the size of gene is 2283 bp ,so i separate it into two parts (each part about 1.5kb) and thinks to ligate them to get a full length.
i can easily get the C' terminal part,but not with N'
i've tried lots of methods,like : change primers;change annealing temp.;change polymerase (Hot start, Amplitaq gold, GC rich);adding DMSO (2% ,5%,10%) or BSA or Mg2+......but!!! still, i can`t get the product i want
anyone's advice would be appreciated !
P.S this gene is on the list of MGC clone (100% match) ,but my boss wants me to clone it myself.....i've been trapped for half year 
Are you getting aspecific product or just nothing at all?
Have you tried to amplify the entire gene in one PCR, it should be possible, I routinely PCR 4 kb...
No hairpin? Try to check the secondary structure of your oligos and if there is no strong primer dimer. But I think the best way it is to amplify the entire gene
Have you tried to amplify the entire gene in one PCR, it should be possible, I routinely PCR 4 kb...
I always get aspecific product. The primers I used were designe by Mac Vector (a program) ,theoretically there shoud be no problem with primer dimer or hairpin structure.
Can you tell me how to amplify a long distance gene?(protocol and kit)
Our lab routinely amplify genes that are shorter than 1kb.
Thanks a lot!!
Yeah , no hairpin. I've used four pairs of primers, didn`t work, always get nonspecific product.
Can you give me some advice about improving the specificity or amplifying long distance gene? I have no experience in amplifying a gene longer than 1.5kb><
thanks anyway!