Are there any special rules for primer design? - (Jul/18/2007 )
Any tips or references?
Hmm... I think there are alot to learn from primer designing. Just google some of the websites.
Usually about temperature, high GC content, can't be too long, specificity etc.
i like the MWG recommendations : http://ecom2.mwgdna.com/improve_results_se...ve_results.html
you click on primer design (left panel) and it includes a tm calculator
primer3 is a good software to help in designing primers. its available online.
first of all try to get some help from someone who has designed primers at least once before, if only to double check your final pair, then watch out for these:
1. the primers should preferrably end on a GC clamp, or a series of G's & C's so that it can attach securely to the DNA
2. no rows of T's or A's which weaken the primer's hold on DNA
3. high GC content (higher than 60%)
4. it shouldn't be self complementary, i.e. CAT GCC *GTT* GGC ATG will just fold over on itself on either side of the GTT and form a hairpin loop instead of attaching onto the DNA.
With the primer pairs you picked, you should consider also the following two steps:
1. Check their quality using Netprimer. It helps to find all kinds of secondary structures, even cross dimers.
2. DO A BLASTn SEARCH with your primer pair. No need to use them, if you end up with five alignments in different genes...