mutant PCR - (Jul/06/2007 )

I am trying to do SDM (site directed mutagenesis) of a gene in a pblue script vector (A to G substitution). I also wish to introduce a silent mutation downstream to this mutation to introduce a restriction site (AccIII). This is about 10 bases away from the first mutation. The second mutation is basically to check for the clones I obtain after the mutant PCR (of the whole plasmid) is done, before they are sequenced. I also intend to use PfuUltra and the Strategene quick change kit to create the mutations.
Queries:
1. To introduce both these mutations, is it a good idea to have BOTH these single base changes in both the set of mutant primers? (The mutant primers are complementary to each other except for the mutant bases.)
2. Where in the mutant primer should the altered base be ideally positioned? What is the optimum length of the primer and its Tm?
3. Are there any other specific guidelines to follow to construct these mutant primers? Could anyone please suggest any programs that are available online apart from PrimerX which will help me design these mutant primers? When I seem to even slightly alter the primer specifications, I get no primers generated in the PrimerX program.
PLEASE HELP!!!!


CD

as possible include one mutation in one primer ..if more there is afear of misbinding of primers. ideally include 14-18 bases on each side of the mutation needed., acc to the formula 2a+4c tm above 68 is better.i read that some authors ,made primers with not 100% matches(not complemntery at all length) and they reported that no problem.actually as possible gc%is 40-50% .try to make the ends of primer with g or c and if possible check the next base to your primers and as possible try to end with c or g and the next base in the sequence is a

Thanks a lot for your suggestions, will put them to use immediately.
CD

1. if the second mutation is to check for the first by restriction, I would put them in ONE primer (otherwise you can't be sure to have the first mutation in all the clones you can digest with AcIII).
why are your primers not complementary for the mutation? don't understand that....

2. mutation should be in the middle of the primer, with 10-15 bases on both sides... I also do my mutation with Stratagene QuikChange and alway design my primers accirding to their guidelines in the mannual... their give you a formula after which you can calculate the Tm of your primers

3. we never use a programm, sorry, can't help you...
but it's actually easier to design mutant primers by your own, have a look in the mannual.
it always works fine for us...

in general it says:

- must contain mutation
- should be between 25 and 45 bases in lenght

- melting temperature Tm over 78°C, calcutaed with:
Tm = 81,5 + 0,41(%GC) - 675/N - % mismatch
- N: primer length in bases
- Values for %GC and %mismatch are whole numbers

- Desired mutation shuold be in the middle of the primer with 10-15 bases of correct sequence on both side
- should have a minimum GC content of 40%
- should terminate in one or more G or C bases
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sorry, the primers are complementary for the mutation as well....