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Mutagenesis not working - no colonies (Oct/23/2006 )

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@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.

-britzelbeere-

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.

-phage434-

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.

-britzelbeere-

QUOTE (britzelbeere @ Oct 27 2006, 10:34 AM)
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.


Hi britzelbeere,

I'm also doing some site-directed mutagenesis on a trans-sialidase and I'm having trouble with the Stratgene protocol too mad.gif . 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!

Sara

-sara.pl-

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" mad.gif

If you are using Stratagene, try retransforming more ┬Ál into your competent cells. This worked for my colleagues who have used the kit.

-britzelbeere-

[/quote]
Did you try zero_blunt topo cloning kit? rolleyes.gif

-emouchet-

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