pcr for gene fusion

[mrudulamariam]

Hi ,
i want to fuse a 1 kb DNA fragment with a 600bp one and then clone this chimeric gene in a vector.I have heard that instead of the conventional restriction digestion, ligation method PCR can be used to get the two fragments fused inframe. Can someone tell me how to go about it?

[Ammie]

Hi! I have done that some times!

It has been very easy for me.

I have just made a PCR mix with the f-primer for the first segment and the r-primer for the second fragment and have added about 1 ul of each pcr-product into the tube.

The PCR -program I used was the "usual" one. (I used Pfu Ultra and 95- 2min, 95- 30 sec, 58- 30 sek, 72- 1 min/kb or 1 min when <1 kb, repeat 30 times, 72- 10 min)

I got some bands of the wrong size, but I think it is because I did not purify my products to get rid of earlier primers. But this what not a problem for me because I anyway wanted to cut it out and gelpurify it before I cloned it.

See that you wrote this several month earlier but I will post my answer anyway...

[alichem]

hi
I a malso working on fusions PCR and it works very well.
I amplified the two fragments ..gel purified and then used one 1ul fro meach frangment in 20reaction. with out adding primers I used it for 15 PCR cycles the nafter 15 PCR cycles I added the F primer for first fragment and R primer for hte reverse fragmnets and again used 25PCR cycles.IT worked very well but I also got some unwanted bands..any way I cut and gelpurifed the desired bands usinf zymo kits.

best of luck
Ali