i need to desing the primers for the production of a recombinant protein, any advice??
It depends on what you're doing, really.
There are great programs to help with the process. Primer 3 is an excellent primer design software (http://bioinfo.ut.ee/primer3-0.4.0/), and the primer properties can be checked using Oligocalc (http://biotools.nubic.northwestern.edu/OligoCalc.html), IDT's OligoAnalyzer (https://www.idtdna.com/calc/analyzer), or AmplifX, which can also run in silico PCR (http://crn2m.univ-mrs.fr/AmplifX?lang=en). Finally, the digestion patterns of an amplicon can be checked using NEBcutter (http://nc2.neb.com/NEBcutter2/).
However, if you're designing adapter primers for old-school conventional RE and ligase cloning you're probably going to have to design them 'by hand'. I don't know of any programs that are clever enough to design a cloning scheme de novo.
My honest advice is not to use a programme. Primer design is really not as difficult as some people make out, you just have to be really precise.
In a lot of ways it's easier when you're cloning a specific gene because you don't have a choice about where your primers should anneal - you need the start and end of your cDNA!
Start by choosing the restriction sites you want to use to insert your PCR product with and check they do not appear in your cDNA sequence. Then start building your oligos with an extra 3 nts on the 5' end to give the restriction enzymes room to bind.
nnnRestrSitennnATG......
nnnRestrSitennnTTA.....
Remember to alter the stop codon on the reverse primer if you want a fusion protein with the tag on the c-term. Don't be afraid of long primers if you need to avoid high-GC sequences, modern polymerases can deal with this no problem. I routinely use primers that are 25 to 30nt with Tm of around 72C or 74C. I do the PCR for this with Phusion with annealing at 65C and it works every time.
Aim for a GC content of between 40% and 60%, especially in the last 15nt if it's a long primer. Don't get too concerned with avoiding self-dimers etc, I've never found it to be a huge problem. If possible, try to end your primer on a G or C (but don't go over-board with it, don't end on GGGGGG e.g.).
Once you have your primers designed check that it is in-frame with any fusion-tags buy cutting and pasting between the restriction sites and seeing if you get read-through from your ATG.
And that's about it really. Good luck :-)
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