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Blunt-ends ligation reversing; therefore NO colonies ;(


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#1 Owe2

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Posted 22 April 2014 - 02:34 AM

Dear all,

 

I am making genomic DNA library. I fragmented genomic DNA using Sonication and used DNA blunting kit to prepare fragments for ligation. On the other hand, plasmid vector was digested to generate blunt ends using one enzyme (SmaI) and dephosphorylated using CIAP.

 

Vector: Insert ratio's of 1:1 to 1:5 were tested and analysed on 1% agarose gel. All ratios worked giving bands larger than linearized vector band.

When any of these were used to transform chemical competent (tested with pUC DNA and give good efficiency at all time when transformation is done) and commercial electrocompetent cells, No colonies are found!

Interestingly, when the ligation reaction is re-analysed in 1% agarose gel, the reaction has reversed, linear vector and DNA fragments smear can be seen on the gel. Ligation is repeated with DNA fragments and linear vector, analysed on gel, all worked but after transformation still no colony. Then ligation is reversed. I have even tried cleaning the ligation reaction by phenol/chloroform/IAA with ethanol precipitation, no colonies and ligation reaction reversed.

 

Have anyone ever experienced this (reversing of ligation)? Or anyone with a suggestion.

 

Now am known as elecrophoresis "dude" because I run these gels like it my profession :)



#2 phage434

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Posted 22 April 2014 - 05:00 AM

Running gels following ligation is seldom worthwhile, in my experience. Just directly transform. I'd suggest that your cells may not be competent. It's the most common problem.



#3 Owe2

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Posted 30 April 2014 - 07:19 AM

The reason I've been running gels was because no colonies were obtained after every cloning and transformation while control pUC19 and control uncut vector were working suggesting that cells are competent.

 

I am suspecting that not both ends of the insert fragments are blunted and as a result only one end can ligate to vector and the other doesn't.

 

thanx



#4 phage434

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Posted 30 April 2014 - 09:41 AM

It's possible. Don't fool yourself by thinking that transforming 100 ng of pUC19 tells anything about competence. You need to try transforming 100 pg of pUC19 and counting the colonies.

CIAP is a poor enzyme, in that it is difficult to remove or deactivate. It can also chew up your DNA. I would switch to shrimp alkaline phosphatase or antarctic phosphatase, both of which can be heat inactivated. I would also use the minimum amount necessary.






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