Help reamplifying the Fusion PCR - (Apr/18/2007 )

Dear all
i have succefully done fusion of two different genes i.e. in the Midle of 1.2kb fragment(for example A) i have inserted 2.44kb fragment(For example (with 600bp of A on each side).i have seen the 3.64 kb band on the gell.
now i am doing the confermatory test. in some of the test i have got good results like when i amplify a fused 1.4 kb fragment with the help of farword primer (some where and A) and a revers primer at the 3'-end of B, and also good result when i used farword primer from 5'-end of B and revers primer some where in 3'-end of A.
but i tried to amplify the full 3.6 kb fused fragment with 5'-end farword primer A and 3'-end revers primer A it is not working.
i will be thankfull if you could help me in my experiment. an idia of my problem is gievn inthe figure bellow;


...................... 5'=========== °°°°°°°°°°°°°°°°°°°°°°°°°°°°°° ===============3'
............................ 0.6kb(A) ........................2.44kb..................................0.6kb(A)
......................................F_________________________________R (Amplification work good)
........................................................................1.4kb
......................................(Amplification work good)F_________________________________R
...............................................................................1.4kb
........................5' ____________________............................._________________________3'
......................................................(Amplification not working)3.64kb
Nafees
Lab. geni.chem
INP ENSAT
toulouse France

Which polymerase are you using for the test? How long is the elongation time? From the diagram, the 5' primer has not been shown to be working, have you used it before in some other PCR? You don't have 3.6kb, but what do you have in the gel when you run the PCR products?

thanks for reply
i am using trying it with two types of polemerases 1. normal invetrogen taq polymerase 2 long expand polymerase.
the elongation time is 4 mints
i did not understand y the 5' is not gouing to be working, for the same fragment once i have used 5' farword primer with a revers primer some where in the 2.44kb fragment and it worked although that has given me two different bands but the actual band were present.
the last time when i run the pcr i have got a smal band instead about 500bp
before that i have got a smear like straight clouds.

Hi,

- If you get smear, maybe too much template, template is not pure enough, annealing temperature is too low... But I assume that you use the same template for other PCRs (is the template DNA from gel or miniprep?) so it should not be due to purity.
- However, the fragment is quite big (~4kb). PCR is quite possibly to have problems. If you just want to confirm, some cheap polymerase like simple Taq (Amersham) should do the trick.
- Furthermore, if it is just to confirm that you have it, the second PCR (it does mean that you can PCR out the inserted fragment using 2 primers designed inside the first gene, right?) should be OK, since it shows the joint part and all the work you have done before are mostly cut and paste, right? Also,
- For the purpose of confirmation, even if you can PCR the big fragment 3.6kb, you still need to do sequencing. And no sequencing can get you the full 3.6kb by one reading, anyway. So design a few more primers, staging inside the construct and sequence it (one direction is fine) together with your current primers. If the sequencing is good, there is the best confirmation (you have to get it anyway before doing anything else).
- The reason I asked about the 5' primer is because I have got problem with PCR a few time due to primers. Since you said it did work before, then fine.

In a nut shell, I think the best way to confirm is just sequence the whole thing. Unless you really need to PCR the fragment out for some other purposes?