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Identifying positive bacterial clones - protocol?

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Identifying positive bacterial clones - protocol?

Postby dakims » Sat Sep 19, 2009 1:48 am

I just transformed heat shock cells with the plasmid of interest hopefully with the insert and I grew them on antibiotic plates to select for the plasmid. So now I have maybe 30 colonies and I was wondering what the next step was. Should I take a tiny piece of a number of colonies and streak them onto individual plates? Or, should I do a little streak on one plate with maybe 8 different colonies (not with the purpose of getting single colonies)? Once I grow them then I do colony pcr or a restriction digest?

the insert is ~2kb
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Postby JackBean » Sat Sep 19, 2009 9:09 am

I guess, you do not have the blue/white system :)

Easiest way is probably to grow them in liquid media (in like 2-3 ml), isolate the plasmids and do some restriction analysis (best would be, if you were able to regognise both, whether you have insert AND whether is your insert oriented in the right way). And if will you get some positive clones, than send them for sequencing.
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Postby canalon » Sat Sep 19, 2009 4:04 pm

I would go for colony PCR, 1 primer in the plasmid, the other in the insert (say within 500bp from one of the end of your fragment) so that you can check presence and orientation at the same time. Test a few clones by dipping a pipette tip in one colony then in the PCR mix (do not count any volume for template, just put water) and you have your results within 2h.
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Re: Identifying positive bacterial clones - protocol?

Postby dakims » Mon Sep 21, 2009 1:29 am

this was using a 5 minute cloning kit and i think its supposed to be pretty efficient. there are primers inside the kit. should i just do colony pcr for a couple colonies?
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Postby canalon » Mon Sep 21, 2009 2:31 pm

That will tell you if the fragment is inserted. If that is all you need to know, yes you can use that. If the orientation is important (because you want to express the fragment, or you want to make modifications for example), you will need one primer in the fragment, and the other in the vector, so that a change in product size will be evident after PCR.
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Re:

Postby JackBean » Mon Sep 21, 2009 5:39 pm

canalon wrote:so that a change in product size will be evident after PCR.


I think the presence/absence will be quite informable ;)
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Re: Identifying positive bacterial clones - protocol?

Postby dakims » Tue Sep 22, 2009 5:56 pm

this a real newbie question but... if you want to do colony pcr, do you have to streak for single colonies? i can't remember how i was taught a while ago but i thought i just got a new plate divided into about 8 areas and take individual colonies from the original plate and just sort of smear (excuse this unscientific language) it on the plate with no intention of getting single colonies. but when i did colony pcr this was to see just to see if there was a plasmid, if i'm not mistaken... then if those came out positive, i did a restriction digest to see if the insert was actually in. Does this sound like it makes sense whatsoever? This is really my second time doing anything and I'm having to figure it out all by myself since the post docs don't seem like they want to help haha
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Postby JackBean » Tue Sep 22, 2009 6:36 pm

What canalon sugests is:
if you need to know presence/absence of insert only (e.g. for multiple re-cloning), you can use primers for the vector, outside of the insert place. The size will tell you, whether there is something or not.
If you need to know also the orientation (e.g. for expression), than you have to use one primer for plasmid (lets say forward) and one (now reverse) for the gene. If there is the gene in good orientation, you will get product of correct size, if is it reversed, you won't get any product, because both primers will work in the same direction.

If you do a colony PCR, than you have to use single colony, of course. The presence of plasmid shall be confirmed already by growing bacteria on media with some selection (antibiotics, presence/absence of nutrition etc.)
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Re: Identifying positive bacterial clones - protocol?

Postby dakims » Wed Sep 23, 2009 1:48 am

thank you all!
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