Fusion/overlap PCR troubleshooting - (Jun/09/2006 )
I am performin a PCR fusion of 3 fragments - a 4kb upstream (fragment 1), 500bp middle fragment (no. 2) and a 4kb downstream fragment. I have carried out the initial PCR of each fragment using primers with a Tm around 68 (high value but needed as specific sequence desired!) and purified each. Decided to fuse two fragments first instead of all 3 but its not working. Used the following
2.5ul of each fragment
4ul Phusion buffer
and appropriate water volume
cycled: 98C for 3', anneal at gradient 37-44, extend for3'20" for two cycles
then added 2ul of appropriate outside primers and cycled at 98C for 30", 65C for 30", 72C for 3', 24 times
no avail however - any tips, bit of a difficult one
I just did a fusion PCR the other day and it worked beautifully to fuse 500 bp frags to a 1.2 kbp marker - not once but twice! I just used normal taq- Promega's GoFlexi's actually supplemented with 1 uL per 50 uL of 10 mg/ mL BSA for luck and 45 cycles. Fusion PCR seems pretty robust - good luck with it
how long is your overlap region?
I have linked several fragments easy, my overlap region is 18 bp. I don't know whether the short overlap cause.
your enzyme must amplify long fragment.