Mutagenesis not working - no colonies (Oct/23/2006 )
@missele: thanks a lot for the advice. Right now, I am the only one who's doing the Mutagenesis. I'll try to adjust the transformation and if that doesn't work, I'll start Plan B next week.
I don't think there is anything special about the cells provided with this kit. I think they are just high competence, normal cloning cells of the type Stratagene normally provides. The genotype is unremarkable for a good cloning strain.
Finally, I got some colonies to pick. But actually the changes I've made in the protocol are now not even similar to what Stratagene recommends.
I'm also doing some site-directed mutagenesis on a trans-sialidase and I'm having trouble with the Stratgene protocol too . I'd really appreciate it if it would be possible for you to post your ammended protocol on here. It would be a big help.
Thanks in advance!
I think I posted in the other thread from Klaas.
Are you using Stratagene reagents??? Cause I didn't and I think that accounts for the "not working part"
If you are using Stratagene, try retransforming more µl into your competent cells. This worked for my colleagues who have used the kit.
Did you try zero_blunt topo cloning kit?