Fusion PCR - Help ragrding Fusion PCR (Mar/08/2007 )

I have problem in fusion PCR of the attached products. can any one help me regarding this problem.
what PCR condition i should use. whch PCR kit and in which combination i should prepare my PCR mix.

i will be thankful if you could help me out in this problem

Bacha

Hi there,
I had the same problem before. I tried to join 3 fragments together (My fragments were 80 bp, 1500 bp and 750 bp) and clone them into a vector as a fusion gene. I tried everything with no success, until I used sequencial PCR to add them together.
First, I added part of the small fragment (80 bp) to the 1500 bp fragment by PCR at the 5' end using primer of 68 mer length. then, I completed the sequence with another primer adding the restriction site at the 5'end. In the first PCR, I used reverse primer with part of the other fragment (750 bp fragment) which has a restiction site that I can use to ligate both fragments together.


after the second PCR, I had a fusion gene consists of the small fragment with a restriction site at the 5' end, the large fragment 1500 bp and part of the 750 fragment which contatins an internal restriction site.
Then I digested both fragments with the internal restriction enzyme and did a linear ligation of both fragments
after gel purification and digested the fusion gene from both termini and ligated it into my vector.

I used Iproof kit from biorad for PCR reaction and primers up to 66 mer.

thanks

Just adding to Anwar's post, sometimes when the joining PCRs aren't working well it's a good idea to ligate the bits together or even more preferably ligate together and into the vector. Sometimes you don't have the sites to do this, but if you can it's definitely a good option. Sometimes this whole cloning business is about exercising a few options so that when one doesn't work the other will.

the most important thing is the melting temperature of your binding sites. Make sure they are all have about the tm, around 58 to 60 Celsius. Avoid self annealing and hair pin structures. Use a tm calculator to decide this

Also considering the size of the products, a proof reading enzyme (eg KOD hifi, vent etc) is required. Basic Taq has difficulty ampilfying fragments this large, and introduces too many mutations.

As already mentioned, while it is possible to synthesis the whole DNA segment at once in one large PCR reaction. It is easier to ligate the fragments in a stepwise manner.

thank you all
i will try this time the way you all have mentioned, and hope that it will work. if i have got any problem i will contact you all again.

thanx