Dne sobota, 25. ledna 2014 16:36:44 UTC+1 Mega [Andreas Stuermer] napsal(a):
That surely is the best bet for small genes.How about this: Use a restriction site that contains ATG? But you would also need PCR to introduce it.
Or a nice restriction site in front of the gene? So your primer is nnnnnggtaccATGnnnnnnn? The a 3 bp before atg seems to be the most important part. gccaccATG is consensus, you might get nice expression levels with GgtACCATG also?
The problem with the restriction site (here NcoI) is, that it also changes the first aminoacid residue after the initial Met because it introduces G as the first base of second triplet. Also there might be another Nco somewhere in the CDS. Thus it might or might not work well enough with the intended CDS. I would really go the PCR way, it is easy enough and works almost every time. It can also be done in paralel with plenty of genes, providing you have enough money for primers. If the cds is really large ~ 10kb plus, you can always do it "per partes" using naturaly occuring unique restriction sites.
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