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Ligation-transformation...Never faced such problem ever

no colonies

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6 replies to this topic

#1 neuron

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Posted 03 January 2014 - 10:25 PM

Hi,

 

I have done lot of ligations, transformations in my previous lab, but I have never encountered such problem, which was asked by a junior at my current place. The cloning that he is doing is very simple- 

 

he has 1kb insert that he cloned in pGEMT vector first, using the kit. he got the clones, and then he cut the insert using the sites BamH1 and Hind III. Now he wants to clone it in pET28a vector to express his protein. He is using JM109 competent cells. Whenever he transforms his ligated mix, he never gets colonies. I told him to try all sorts of things that we generally tell...to check the comp cells efficiency, ligase etc. His comp cells are fine as he always gets colonies with pET plasmid. Ligase also seems to be working as he loaded both ligated product and empty plasmid on gel and he observed a shift in ligated product. I am attaching the gel picture herewith. I don't know what is going wrong. Molecular biology experts, please help to solve this problem.

 

 

Thanks

Attached Files



#2 phage434

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Posted 04 January 2014 - 06:26 AM

The fact that you can transform your "competent" cells with minipreped plasmid tells virtually nothing about how competent they are. You need to measure the competence, by doing serial 10x dilutions of the plasmid to establish the competence, measured in CFU/ug of plasmid. It needs to be at least 10^7 cfu/ug for routine cloning, and it would be better if it were more like 10^9. Your ligations, even very successful ones, produce very small numbers of correct molecules.

 

Your gel shows very large fragments, probably the result of concatamer formation. Likely this is a result of far too high a concentration of vector and insert. More is not better. Aim for low concentrations (around 20 - 50 ng or so of vector in a 10 ul ligation) and equimolar or slightly greater amounts of insert. Concatamers will not transform.



#3 neuron

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Posted 04 January 2014 - 09:00 AM

Thanks Phage, Its a great help!! yes he is using around 80ng as ligation mix. I told him to reduce as you suggested. Hopefully he will get the colonies nowsmile.png . I will let you know. 



#4 neuron

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Posted 08 January 2014 - 07:57 PM

HI Phage, This time 40ng was used for transformation but again no colonies in ligationhuh.png ...I don't know what is wrong, now I am suspecting some problem in vector...what else could it be?mad.gif



#5 neuron

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Posted 08 January 2014 - 08:21 PM

One more thing, he used pRSET for ligation 40ng..there he is getting a bacterial lawnblink.png



#6 phage434

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Posted 09 January 2014 - 11:19 AM

Just to make sure. You are plating these out on Kan plates, rather than Amp plates -- pGEMT is AmpR, while pET28 is KanR.

 

Otherwise, measure the competence! This is THE most common problem. Unless you measure it, and can be sure it is acceptable, you are flying blind. Also, do a no-insert control and a no-ligase control.



#7 neuron

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Posted 09 January 2014 - 07:36 PM

Yes Phage we are using Kan for PET28. What about NEB quick ligation kit? How is that? We are using that kit? In my previous lab we used to do 16 degrees ligation and it used to work fine.







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