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no colonies after electroporation

electroporation bacterial transformation

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8 replies to this topic

#1 sammy_07

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Posted 10 November 2014 - 10:50 PM

I have a construct (received from someone else) which I have to transform into DH5a. But after plating there is no colonies on selection plates. I don't think the problem is with the cells since I can see growth on LB only plates. I did ethanol precipitation before electroporation. What could be the problem?



#2 pito

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Posted 11 November 2014 - 02:12 AM

you did an ethanol precipitation before you transformed? WHy? How did you receive the construct?

You know for sure what the selection marker is?


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#3 sammy_07

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Posted 11 November 2014 - 02:59 AM

The construct is not available commercially and I was able to get it from a lab overseas where they worked with the same one. When I failed to get colonies I thought it would be better if I precipitated the plasmid to increase the concentration but that didn't help too. And yes, I checked the selection marker.



#4 pito

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Posted 11 November 2014 - 03:34 AM

give the protocol.

 

And why not use a standard LiAc transformation?

Do you still have plasmid left? Whats the concentration? How did you receive the plasmid?


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#5 phage434

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Posted 11 November 2014 - 07:03 AM

Almost certainly the problem is low  competence of your cells. I would debug this by using a common plasmid which has the same antibiotic resistance as your plasmid, and measuring the competence of your cells. Alternatively, you can use commercial cells, which are usually highly competent. As has already been suggested, make double sure that you are using the correct antibiotic for selection.

 

http://openwetware.o...competent_cells



#6 merlav

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Posted 18 November 2014 - 05:20 AM

ETOH precipitation is for clean your DNA if it have too many impurities not for concentrate a sample, you lose material with it so use only as the last resource. If want to concentrate a sample just place in a speed vac in a low setting.  The cause of not getting  colonies are:

1. You don't have the proper ratio of plasmid:bacteria

2. That your bacterias are low competence and it doesn't have anything to do of how well it grow in LB.    

3. Incorrect antibiotic. 


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#7 Hic5Prostate

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Posted 18 November 2014 - 09:55 PM

I have never heard of electroporating a plasmid into DH5alpha bacteria.  If you are buying commercial rather than generating your own, you should be able to directly add the plasmid into the bacterial suspension, incubate on ice, and heat-shock it.  You're better off buying commercial DH5alphas, especially when you remember that you can freeze your colonies into glycerol stocks for later reconstitution (and thus do not need to transform over and over again).  However, what is stated above regarding competence is true.  If your plasmid is doing something funny vs any other plasmid, you can check for this by transforming with pUC19 plasmid and plating on an agar plate containing ampicillin.



#8 bob1

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Posted 18 November 2014 - 11:44 PM

I have never heard of electroporating a plasmid into DH5alpha bacteria.  If you are buying commercial rather than generating your own, you should be able to directly add the plasmid into the bacterial suspension, incubate on ice, and heat-shock it.  You're better off buying commercial DH5alphas, especially when you remember that you can freeze your colonies into glycerol stocks for later reconstitution (and thus do not need to transform over and over again).  However, what is stated above regarding competence is true.  If your plasmid is doing something funny vs any other plasmid, you can check for this by transforming with pUC19 plasmid and plating on an agar plate containing ampicillin.

 

Electroporation of bacteria is pretty common and usually gives a higher rate of transformation, but if chemically competent cells are competent enough (commercial preps certainly are) then electroporation shouldn't be needed... i've certainly never had any problems with my home-made chemically competent cells, even ones with poor competency.



#9 phage434

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Posted 19 November 2014 - 06:07 AM

Doing a test transformation with a plasmid with the same antibiotic resistance tests not only your cells, but also checks the plates. If you have problems, this should be your first step.







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