I would like to make an expression vector, and attach a tag to the 5' end, so the start codon will be moved upstream.
My forward primer will contain a restriction site, the kozak (with the START), the tag, and then followed by the first 18-20 nucleotids (without the START) of my coding sequence. The reverse will be built up by the last 18-20 nucleotids of my coding sequence, till the STOP, and restriction site.
Primer planner programs say that these primers are unacceptable, the melting temperatures and other parameters are very bad. (I controlled only the 18-20 bases of the coding sequence, not the whole primer).
So what is better: to try these "bad" primers, OR use other F primer fitting on the 5' upstream region, and R primer fitting on the 3' downstream region?
This will cause some aminoacid plus at the N terminus between the tag and my coding sequence.
thanks
Hi Magdolna,
Unlikely you face any problems with non-specific products if you deal with vector. Even 5 -7 degrees or 3-4 nt of difference in size or Tm between primers is ok.
Hi Magdolna,
I quite agree with Andrey, it is not likely to be that big of a deal.
If the Tm is too low, consider adding a couple extra bases to the 3' end of your oligos (more coding sequence). If too high, don't worry. For this sort of cloning, I often do the first 5 cycles at a lower annealing temperature since only the coding sequence of the oligos match the target. After 5 cycles, most of the target will have the RE & Kozak sequence incorporated. I then bump up the annealing temperature for 30 cycles. Use a good proof-reading polymerase (I like Q5 from NEB). Cloning error-free large genes with regular Taq can be frustrating. I use NEB's Tm calculator for annealing temperatures - it works quite well and has options for many types of buffers & polymerases.
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