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Cloning contamination


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#1 Clare

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Posted 25 July 2014 - 01:20 AM

Hi all,

 

The Cloning Gods have definitely not been with me lately!

 

Everyone in the lab has been having issues cloning too :/ I suspect contamination but how on earth do we go about cleaning things up? I've never had this issue before.

 

I have attached a gel from yesterday where lanes 2, 5-13 contain all sorts of unexplained bands (this is a NotI digest). Colonies were grown on LB Amp plates. I used fresh commercially available competent cells.

 

Any ideas on how to make this go away?

 

Thanks in advance!

 

xxx me and the rest of the lab

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#2 bob1

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Posted 25 July 2014 - 02:36 AM

It would be very very unusual to have a generalized contamination of clonign unless some of your bacterial transformation stocks contain something that they wouldn't normally have.  You could try a plasmid prep on bugs alone without transformation and see what comes out.  If possible go back to some older stocks or borrow some from another lab and try those too.

 

It could also be possible that you have contaminated stocks of ligase or commonly used restriction enzymes (if communal), but it would require significant contamination with uncut plasmid to get much to show through.

 

Basically, if contamination is actually the problem, it should go away if you throw out communal stocks of everything and start afresh.



#3 Clare

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Posted 25 July 2014 - 02:47 AM

Hi Bob,

 

Do you reckon extensive colony PCRs could contribute in any way? I know hundreds and hundreds have been performed in the lab recently.

 

Clare



#4 bob1

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Posted 25 July 2014 - 02:51 PM

I doubt it, the only way they would cause contamination is if you were carrying some of it over into the PCR products you are trying to generate for insertion into backbones. PCRs should only generate shortish fragments, which shouldn't then be capable of being replicated if they did get transformed.






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