As I have started to learn primer designing, I have come across terms like restriction sites, sitting sequences, etc. I wanted to know if sitting sequences added before the restriction sites are specific to the restriction endonuclease or independent of the restriction enzymes and, if at all related, what factors decide a good sitting sequence.
I have not come across a definition for a sitting sequence but if it is just the few random bases added to a primer to allow efficient cutting then any random bases will do but I would avoid adding CC or GG if the digest site contains those motifs so that G quadruplex or hairpin looping do not happen and if adding 4 bases to a 4 cutter do not make the 4 bases the same as the restriction site. The added bases are independent of the enzyme that you are using
I agree with Paul Rutland. I used to systematically add three As to prevent hairpin formation.
There are several reasons to design primers. In case you're gona use them to clone a gene into a plasmid. First, you need a restriction site to Create blunt or sticky ends, so you need to choose the specific sequence according to the restriction enzymes you desired. Knowing you're going to remove the end site, you can add AAA or GGC or non complementary sequences to avoid primer dimers. No commonly, but if you want to design primers for gateway method, you will need specific sequence at the end according to the organism (plants, mammals, yeast).
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