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molecular cloning troubleshooting


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#1 yii

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Posted 02 June 2010 - 10:41 AM

Hi everyone,

I'm student in internship in a laboratory in biomolecular technology.
I have to clone two proteins.
To do that, I've made a PCR from ADNc library to extract my ADNc in agarose electrophoresis then I've done purification. After ligation, transformation, minipreparation ... my positive miniprep have been sequenced and surprise, bacterial inserts and no interests proteins !

I'm trying to understand which step could cause contamination of my inserts ! extraction ligation, transformation in top10, minipreparation ...

Anybody has got an idea ?

#2 bob1

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Posted 02 June 2010 - 04:15 PM

I would suspect the miniprep first - make sure you stick closely to the times for the lysis and neutralisation steps, also make sure you invert the tubes gently, not vortex or pipette, during these steps.

#3 HomeBrew

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Posted 02 June 2010 - 05:21 PM

I have to clone two proteins.


You need to be a bit more careful with your terminology -- you're swapping between DNA and protein terminology. You can't clone a protein -- you can, however clone a gene that encodes a protein.

I assume an ADNc library is a cDNA library? What do you suppose the source of the bacterial DNA inserts was? Have you tried sequencing the amplicon produced by your primers (without cloning -- just send the gel-purified band for sequencing)? If the PCR product is not what you're looking for, then you need to re-design your primers to be specific for your cDNA. It's likely your primers are amplifying something from the chromosome of the bacterial host holding your cDNA library.

PS. It's possible I've misunderstood what you're saying happened -- if so, please try and explain it again.

#4 yii

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Posted 03 June 2010 - 04:02 AM

I have to clone two proteins.


You need to be a bit more careful with your terminology -- you're swapping between DNA and protein terminology. You can't clone a protein -- you can, however clone a gene that encodes a protein.

I assume an ADNc library is a cDNA library? What do you suppose the source of the bacterial DNA inserts was? Have you tried sequencing the amplicon produced by your primers (without cloning -- just send the gel-purified band for sequencing)? If the PCR product is not what you're looking for, then you need to re-design your primers to be specific for your cDNA. It's likely your primers are amplifying something from the chromosome of the bacterial host holding your cDNA library.

PS. It's possible I've misunderstood what you're saying happened -- if so, please try and explain it again.


Sorry for my terminology, I'm not a great english speaker ! But thanks for your answers.

Yes, I've sent my cDNA extracted from my agarose to sequencing, I hope it will be good.

Minipreparations can contaminate my clones, but how ? Pipetting ?

First, I believed that it was my pCS2-HA plasmid, could it be possible that a bad ligation between plasmid vector and inserts can produce a sort of recombinaison with bacterial plasmid of my top10 E.Coli ?

Thanks for your help!




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