overlap PCR - (Sep/28/2006 )
Currently i'm trying to do an overlap PCR to conjugate my protein of interest to EGFP, but my protein is a big guy which has a 4.5kb, so how long is the overlap region should be9for primers), and anything important for considerations?
Are you trying to create a GFP fusion to your gene? If so, you can find a vector where you can PCR clone your gene in frame to GFP as either a N-terminal or C-terminal fusion. You would have to add the proper restriction sites to the ends of your primers and make sure the final sequence is in frame. If you may also need to delete the stop codon of your gene to prevent translational termination before translation of GFP. Most people also sequence the final gene to verify there were no mutations introdced during the PCR.
i encountered a problem, i tried to combine a 4.5kb fragment with a GFP tag which is about 800bp, but the pcr product i got turned out to be aroud 3kb. I use a his tag as the overlapping region( which is right after my 4.5kb pcr fragment and in front of gfp), so when i put the two fragments together i set the annealing temp at 50 deg and 52 deg but both gave me the 3kb band, shall i drop the temperature more?
This technique should be possible for you - be sure to choose an appropriate polymerase for long, error-free amplification. Attached is a report using a similar technique but intended for generation of c. elegans reporters. They describe some useful steps which may help you.
Hi,thx for your info. On my gel, there are several bands and one is suspected the right one(about 5.3kb), however, after i did the gel extraction and PCR again, i still have the problem of getting the correct-sized band. It keeps giving me a weird 3kb band and a 800bp band which probably is the GFP. Any idea, it really freaks me out.
may be u still have to increase the temperature to avoid wrong primer annealing