Hi all. I am trying to fuse 2 fragments into 1 using overlap extension PCR. The lenght of the fragment 1 around is 3kb and fragment 2 around 300bp. All 2 fragments are derived from previous PCR reactions and have been cleaned using the PCR cleanup colums. I am using Taq polymerase for this matter.
I am doing the overlap extension PCR in 3 steps, the first one being with primers, the second without primers (15 cycles, 76ºC), and the third one with the addition of the outer primers and an appropriate Tm in the annealing stage. I have been able to get the fragments of the correct size after the first PCR, but not in the final stage. After the second PCR where i added the primers it seems that the primer is binding somewhere else before the desired lenght (2kb). I've tried to change the Tm for the final step as well as designing a new primer, but nothing changed. Any tips in how can I improve it?
It might help to isolate the problem primer. So if you use the 3kb amplimer as the dna template and run all possible primer combinations in a pcr ....3kbf and 300f, 3kbf and 300r, 3kbr and 300f, and 3kbr and 300r then if any combination gives your unwanted band you will know which primer to redesign. Sequencing the 2kb fragment might also give a mechanism for what is happening.
If the primer isolation experiment fails then it may be that the addition of the 300 base fragment causes a loop of 1kb in the 3kb template and the taq is reading across the neck of the loop so amplifying a smaller product. In this case try the 3.3kb pcr in the presence of a secondary structure reagent like final concentration of dimethyl sulphoxide (DMSO) at up to 8% dmso
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