Site-directed mutagenesis - (Jun/15/2007 )
Hi there
I'm trying to do site-directed mutagenesis.My vector is puc18 and my insert is a 504bps fragment of DNA.
I did double digestion and cloned that 504bps in my vector. Now at first I should design my primers. I have 2 cysteine that Im gonna change them to the Alanin ( TGC to ).
There are 2 cysteine with 15 bps between( ...TGC...............TGC....).Im gonna change those to (...GCC............GCC...).
Can I do that with just one primer to make those changes? is it possible?How would be my primer?? If not can I design one 2 primers with just doing one PCR??
I'm trying to do site-directed mutagenesis.My vector is puc18 and my insert is a 504bps fragment of DNA.
I did double digestion and cloned that 504bps in my vector. Now at first I should design my primers. I have 2 cysteine that Im gonna change them to the Alanin ( TGC to ).
There are 2 cysteine with 15 bps between( ...TGC...............TGC....).Im gonna change those to (...GCC............GCC...).
Can I do that with just one primer to make those changes? is it possible?How would be my primer?? If not can I design one 2 primers with just doing one PCR??
Hi,
I had applied this procedure before on M13 SD152 platinum binding peptide C7C region. and in my case, I also had applied double mutations. I firstly applied the first mutation using one set of primers and after obtaining the mutated template, by using second set of primers, I had completed the experiment.
good luck and I hope this helps
I'm with MuratA on this one. Use two separate primers (if possible); each one in a different reaction. Using one primer means you will either have a very, very long primer which will not work or you will have the mismatches at the end of your primer meaning your replication will be a mess.
There are some good primer design programs out there, but most of them are expensive. Though, I have successfully developed site-directed primers using PrimerX, an online program. It can be found at: http://bioinformatics.org/primerx/
Seriously, don't forget to treat the DNA with Dpn I to get rid of the parent plasmid. The manual for the QuikChange® II Site-Directed Mutagenesis Kit (Stratagene, cat #200523 or #200524 -- 10 or 25 rxn respectively) has some extra tips for SDM reactions (please note that I do not have any experience with the kit itself, nor do I have anything to do with Stratagene besides as a user of their products). The manual can be downloaded here: http://www.stratagene.com/tradeshows/feature.aspx?fpId=142 (if it works directly).
Good luck!
I'm trying to do site-directed mutagenesis.My vector is puc18 and my insert is a 504bps fragment of DNA.
I did double digestion and cloned that 504bps in my vector. Now at first I should design my primers. I have 2 cysteine that Im gonna change them to the Alanin ( TGC to ).
There are 2 cysteine with 15 bps between( ...TGC...............TGC....).Im gonna change those to (...GCC............GCC...).
Can I do that with just one primer to make those changes? is it possible?How would be my primer?? If not can I design one 2 primers with just doing one PCR??
Hi,
I had applied this procedure before on M13 SD152 platinum binding peptide C7C region. and in my case, I also had applied double mutations. I firstly applied the first mutation using one set of primers and after obtaining the mutated template, by using second set of primers, I had completed the experiment.
good luck and I hope this helps
yeah.. easy job..
Design the primer of 25 bp containing both mutations.. pcr it.. digest it.. n ligate in to your expression plasmid..
If you want it really fast.. 1 day job..
use.. stratagene site directed mutagenesis kit... it will amplyfy whole plasmid in PCR.. n you can trasform the pcr products directly into bugs after some purification..
all the best..
[...]
use.. stratagene site directed mutagenesis kit... it will amplyfy whole plasmid in PCR.. n you can trasform the pcr products directly into bugs after some purification..
[...]
So you had good results doing this with the Stratagene kit? No mismatches, no problems with loose overhangs of the primer, etc? Interesting... I should check this kit out.
[...]
use.. stratagene site directed mutagenesis kit... it will amplyfy whole plasmid in PCR.. n you can trasform the pcr products directly into bugs after some purification..
[...]
So you had good results doing this with the Stratagene kit? No mismatches, no problems with loose overhangs of the primer, etc? Interesting... I should check this kit out.
Never had a Problem.. except money.. it took me whole month to convince my boss.. coz i hod to make more than 15 mutated clones to make for binding analysis.
http://www.stratagene.com/manuals/200518.pdf
but few months back.. i use overlap PCR.. which also worked perfectlty.. its very cheap.. i always use normal Taq.. lol
[...]
use.. stratagene site directed mutagenesis kit... it will amplyfy whole plasmid in PCR.. n you can trasform the pcr products directly into bugs after some purification..
[...]
So you had good results doing this with the Stratagene kit? No mismatches, no problems with loose overhangs of the primer, etc? Interesting... I should check this kit out.
Never had a Problem.. except money.. it took me whole month to convince my boss.. coz i hod to make more than 15 mutated clones to make for binding analysis.
http://www.stratagene.com/manuals/200518.pdf
but few months back.. i use overlap PCR.. which also worked perfectlty.. its very cheap.. i always use normal Taq.. lol
n on the question of mis match n all... hmm.. i don't think so..
if you have more than 8bp pof primers.. the probability of missmatch is very less.. in your case if you design pcr primers of 25bp.. probability should be NIL.. so i'll not worry about that..