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Introduce restriction sites with PCR primers - (May/25/2007 )

I want to introduce two restriction sites in the end of my insert using PCR primers.
The enzyme need some extra nucleotides to be effectiv on the sides of the sites.

The enzymes are NotI and KpnI.

I have never done this before but I have read that 6 bp are good to add by default. I looked in the appendix in new england biolab catalogue and it argued that KpnI needs two base pair, but they also said that I should add 4 base pairs extra (because they do not know if the single-stranded nucleotides from the first cutting matters).

So is it ok to use 6 extra nt's you think? And does it matter which nt's it is? I have read both that you should us GC and not use GC (because it effects the meltpoint more).

How would you have done it?


I have two suggestion and they have Tm at 80 and 76,7, and that is high... but I suppose it has to be when you make it so large. So how will it work together with my other primers that have a Tm around 60?


We usually add 5-6 bases beside the restriction site. I add A's or T's .