This is my methods: Firstly, I amplify two fragment respectively, after gel purification of two fragments, a fusion step is carried out which only add two fragments, pfu polymerase, buffer and dNTP for about 5 cycles.
95℃ 20s
55℃ 20s
72℃ 3min
then 3 micro liter fusion product will be taken as template for whole PCR amplification.
However, the final gel result is terrible, the gel is almost completely white. I have tried several times, I am sure the quality and quantity of two fragments are fine and overlap has 40bp more or less. The length of two fragments are 1480 and 4000bp.
Anyone can help me about this problem?
Is your "fusion" step a PCR with primers specific to each fragment (ex forward primer anneals fragment #1, reverse primer anneals fragment #2)?
Dear Cristin Healy, I didn't add any primers in my fusion step actually, after getting my fusion products, I took 1 micro litter as template and mix forward primer of fragment 1 with reserve primer of fragment 2.
I have worked on fusion of fragments as well. For this, I do an amplification of each fragment on its own, gel extraction then do a second round of PCR using a mix of the two fragments as the DNA template with the primers " forward primer of fragment 1 with reserve primer of fragment 2". If you're using "pfu", I suggest you could increase the extension time to 5 minutes.Also, you could use another enzyme as "Phusion", the results may be better. Hope this works for you.
@ Nadia Assal thanks for your suggestion, I have already tried different annealing temperature. The fact is that I can get my targeted band on gel now, but there are lots of non-specific bands here, so I purified band from gel and re-do PCR, however smear again. It is so wired.
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