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How to get a mutant in Streptococcus aglactiae - (Feb/28/2006 )

I have my insert in pGEM (the first step is already done), so I lyse with Eco-Bam. Now, I tried to put my insert in pG1 and transform E. Coli (This pG1 is interesting because it has a Gram positive sequences). But I cannot have a E Coli with pG1 (I select with antibiotic of course).

My protocol to transform E. Coli:
Ligation product in DH5alpha competent
45min in ice
10min at 37C
add 1mL TS (already hot) and 1h30 at 37C
Centrifuge 3min Vmax
pull off 200microL of surpernatant
Put 100microL of pellet

Have you idea to try with an another plasmid ou suggestion about protocol?

-AlexCochin-

is this a heat-shock method??

I know that there are many ways to do things, but 10 min at 37???? holy cow that seems like a lot...can you tell me the source of your protocol?

likewise, do you have positive and negative controls, and what are their results?

-aimikins-

QUOTE (aimikins @ Feb 28 2006, 06:18 PM)
is this a heat-shock method??

I know that there are many ways to do things, but 10 min at 37???? holy cow that seems like a lot...can you tell me the source of your protocol?

likewise, do you have positive and negative controls, and what are their results?


It 'heat shock method that's why we put the cellsat 37┬░Cduring 10min.
I will make control (good idea thanks)...

-AlexCochin-

How did you make your competent cells? After your heat-shock step (which I do for 40 seconds at 42┬░C), why don't you just plate all your cells?

-HomeBrew-