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How to know the plasmid extracted are indeed the plasmid of interest?

plasmid e.coli transformation antibiotic selection

Best Answer Trof, 18 January 2015 - 09:32 AM

You need to find what your plasmid is derived from, the sequence would have some similarity.

 

This vector designation is found nowhere on Google. But you can't work with a completely unknown vector. Ask the person who gave it to you or search citations about it (probably under different name), even the old vectors without complete sequence have at least a restriction map. 

 

I would not work with a vector I know absolutely nothing of, that is just so very problematic on many levels.

Last time I was working with someones plasmid, I got different restriction patterns than sequence predicted and finally it was found the vector recombined some years ago, without anyone noticing, just because no one checked it. The sequence they send didn't fit.
So, if you value your work, just refuse to work with a plasmid unless someone gives you the info about it.

Someone must know.

In the mean time, you can use even a spare information on a part of the sequence to design a PCR product to check. Is the vector containing some kind of specific insert you know? You can use a primer in the know insert and some primer on usual vector sites - promoter primers, M13  primers, anything to check you jave your insert in the vector backbone.

 

But as I said, if the vector is supposed to have some other features than insert and antibiotic resistance, you can't be sure it contains them all fine, unless you make at least several restrictions.

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#1 Meg P. Anula

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Posted 17 January 2015 - 08:01 AM

Hi all, I've some problem with my transformation. When I did antibiotic selection, both transformants (e.coli with plasmid of interest) and control (without plasmid) showed growth.

 

I picked a few colonies from the transformant plate anyway and growed in broth with 100ug/ml erythromycin overnight. Then I extracted the plasmid and run agarose gel and the results showed band around the similar bp of the plasmid of interest.

 

Since the antibiotic selection failed (I doubt if the antibiotic concentration is too low), I cannot be entirely sure the plasmid transformation was successful. 

 

So my question is, based on the agarose results, can I be sure the plasmid extracted is indeed the plasmid I want? Or could it be the naturally occuring plasmid in e.coli strain w3110IQ instead?

 

Pls, if anyone has any idea, kindly advice. Thanks.

 

 



#2 CPRES

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Posted 17 January 2015 - 09:04 AM

Quick answer to main question, I am not going into all antibiotics selection business here at this time:

1. You can sequence the plasmid.

2. If you have the plasmid DNA sequence, you can design restriction digests that would give you unique and expected size bands.


So. Now that you have your first ever question on bioforum answered (or not), mail yourself your username and password so you don't forget them, and then come back soon to update us on how it all worked out. That's how you build Karma in science.


#3 Trof

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Posted 17 January 2015 - 11:55 AM

As mentioned.

Due to the usual plasmid sizes, restriction with several RE and comparing the restriction patterns and sizes with the expected ones is used or PCR a region specific to plasmid/vector. 

 

I never heard of a "natural" plasmid appearing in a competent cell stock, but in any case you need to be sure that your plasmid is intact before proceeding with work. 


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I never trust anything that can't be doubted.

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#4 Meg P. Anula

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Posted 18 January 2015 - 07:32 AM

Dear CPRES and Trof,

 

Thanks for the response.

 

I do not have the plasmid sequence. I'm using psGGAC332 plasmid and the sequence is also inaccessible online.



#5 Trof

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Posted 18 January 2015 - 09:32 AM   Best Answer

You need to find what your plasmid is derived from, the sequence would have some similarity.

 

This vector designation is found nowhere on Google. But you can't work with a completely unknown vector. Ask the person who gave it to you or search citations about it (probably under different name), even the old vectors without complete sequence have at least a restriction map. 

 

I would not work with a vector I know absolutely nothing of, that is just so very problematic on many levels.

Last time I was working with someones plasmid, I got different restriction patterns than sequence predicted and finally it was found the vector recombined some years ago, without anyone noticing, just because no one checked it. The sequence they send didn't fit.
So, if you value your work, just refuse to work with a plasmid unless someone gives you the info about it.

Someone must know.

In the mean time, you can use even a spare information on a part of the sequence to design a PCR product to check. Is the vector containing some kind of specific insert you know? You can use a primer in the know insert and some primer on usual vector sites - promoter primers, M13  primers, anything to check you jave your insert in the vector backbone.

 

But as I said, if the vector is supposed to have some other features than insert and antibiotic resistance, you can't be sure it contains them all fine, unless you make at least several restrictions.


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#6 CPRES

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Posted 18 January 2015 - 04:10 PM

Not doubting you, Meg, but are you sure the name is not pCAGGs.. That is a more common plasmid backbone. Anyways, otherwise I would listen to what Trof said.


So. Now that you have your first ever question on bioforum answered (or not), mail yourself your username and password so you don't forget them, and then come back soon to update us on how it all worked out. That's how you build Karma in science.


#7 Meg P. Anula

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Posted 19 January 2015 - 07:09 AM

Trof, 

 

Thanks for the explanation, it's very useful.

 

CPRES,

 

Thanks for noticing the typo. It's psGANC332, a plasmid from lactic acid bacteria.

 

 

I do not have the complete sequence but I do have the restriction map, I'll do as suggested by Trof to confirm. 

 

I am truly grateful and appreciate your response :)







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