Hey, I am designing some primers for a cloning. It turns out that I want to use a hexaglycine linker in order to produce fusion proteins, and in order to do so I have to use a primer that would look like : GGTGGTGGTGGTGGT GGTxxxxxxxxxxxxxxxxxxxx.
The question is, is this primer safe? Checking the GC content regarding the GGT tandem repeat array it is not so bad (roughly over 60%), but I fear that I might get secondary structures due to homology and thus hamper the PCR efficiency.. Yet I am not really sure. Do you think this primer would be safe? is there any tool I can use to verify it myself?
IDT DNA has primer quest and scitools that you could use to evaluate the hairpins. You should not get self dimers just because of the tag but it depends on your 3' sequence. Make sure you don't have a CC repeats at the 3'. Also try to have a delta G of -5 to -7 on your 3'.
Good luck
I think you can change 2-3 GGT (Gly) to GGA (Gly) and then do a test. I ever did the similar cloning and did not meet the problem.
I have evaluated the secondary structures of your primer (GGTGGTGGTGGTGGT GGT). I did not found any secondary structures in the primer.
I am not sure.This may also need experiment to confirm, the theory may not conform to the actual. I only can sure, using the DNA MAN software, I did not found any secondary structures in the primer.
hey, thanks to everybody! I will try it and post the results when i get them..
- Badges
- Science method