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Inserts too small after transformation


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3 replies to this topic

#1 rpmi

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Posted 13 March 2009 - 06:11 AM

Hi all!
I have cloned a 5kb insert of a viral genome in the TOPO-TA vector. Transformation and blue/white screening reveals 100+ colonies white and blue. A colony PCR using M13 primers (located in the vector) shows inserts in all colonies checked but the fragment lengths differ from 0.8 to 1.8 kb instead of the expected 5.3 kb. Sequencing shows that the inserts indeed come from the viral genome I cloned; the bacteria (Top10 cells) probably kicked out randomly part of the viral genome. I tried the same cloning and transformation strategy in another vector: pUC18 but no luck either. Both vectors used are high copy plasmids: do you think a switch to a low copy plasmid would help? Or should I try another bacterial strain for transformation? Or ...... any suggestion is welcome.

Thanks! Rene

#2 lotus

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Posted 13 March 2009 - 11:31 AM

Are you sure you are actually transforming a 5 kb insert into your bacterial cells? maybe the difference length happens upstream in the cloning procedure.
There must be something unique in your insert sequence that the cells don't like at all. You could look at this aspect and delete the offending sequence. Or maybe gateway cloning (invitrogen) might be worth a try.

#3 rpmi

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Posted 15 March 2009 - 11:08 PM

No, I am not 100% sure about the 5 kb. I could check my ligate though. I cannot clone a shorter fragment due to the fact that we want to have a full genome viral clone.
I have never heard of gateway cloning but I will have a look at it.

Thanks for the reply!

Rene

#4 memo

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Posted 17 March 2009 - 07:34 AM

Clean up you insert before ligation and be sure your transformant selection is done properly. Using cleaned up PCR framents for cloning I always get a lot more white cfu's than blue ones using my own protocol!




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