Posted 10 December 2004 - 04:44 PM
I am trying to clone a gene of length ~5kb. First, I tried to do PCR amplification of the same and din't work. So I decided to amply shorter fragments by PCR method. Now I got 4 different fragments with a sharing sequence (~200-300bp) between the adjacent frangment. The idea was to ligate the fragments using the sharing sequence region. so What I did was: I mixed two fragments (equal conc.) and denatured (94C, 5min) and allowed to cool slowly at RT (10min) assuming that the fragments would anneal in all possible combinations. After that I tried to end fill using klenow's fragment (37C, 30min). The mix had dNTPs also. Then I did PCR amplification using forward primer of the first fragment and reverse primer of the second fragment (Ann. temp. -50C-56C). The expected length was ~2.2kb. Unfortunately I din't get any band of that lenth. The Tm diff between the two fragments was high (13). That may be one of the reason I din't get the band. Or might be because of bad annealing. If anyone can suggest me soemthing for my problem, it would be of great help since I have to do this annealing three more time sothat I would get the whole 5kb gene. I really appreciate your help. Thanks a lot.
Posted 10 December 2004 - 09:56 PM
if an unique RE site is present within the overlapping region(but not in orther parts,so you need to check carefully ),why not try a cut-and- ligate approach?
hope it helps!
God helps them who help themselves!
Posted 12 December 2004 - 10:56 AM
Well, about getting the 5kb full length. I have cloned ~1.2kb long fragments and now I am trying to link them one by one and to get the full length. I tried the full length amplification, but I din't work out. I tried 5' and 3' RACE kit, but for some reason I could get only 1.1kb fragment. So I have to do it a tedious and lengthy way, sothat I could get the full length. But I thought about your idea and I am going to try that too. If you have any other suggestions, please let me know. Thanks a lot!