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Unsuccessful self-ligation


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#1 Eureka

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Posted 22 March 2011 - 02:24 AM

So, I ran out of patience and ideas...
I cut a pCMV-AC-GFP vector (OriGene) with AsiSI (SfaAI) and MluI to remove the cDNA cloned in the ORF (I'm trying to make an "empty" vector to use as a control for the over-expression of this cDNA). Then, I dephosphorylated, filled in the ends with Klenow, and ligated with T4 DNA ligase (using PEG4000 to increase the efficiency of blunt-end ligation). This mixture was used to transform DH5a chemically competent cells which were later plated on LB agar Amp, but I didn't obtain a single miserable colony.
The double digestion controls were fine, the ligation control as well, and the transformation efficiency of the cells is good too. Any suggestion?

Thanks in advance ^^

#2 dpo

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Posted 22 March 2011 - 04:25 AM

eh, why do you dephosphorylate? You need a phosphate group on one of the strands to ligate to fragments together. In a ligation with an insert, the phosphate is provided by the insert, but here you only have one fragment (your vector), so dephosphorylating it will make it impossible to self-ligate (which is actually the reason why you do it when you would want to use this vector in a ligation with an insert). So, briefly, just cut with your enzymes of choice, fill in the ends with Klenow and then ligate.

#3 Eureka

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Posted 22 March 2011 - 04:45 AM

eh, why do you dephosphorylate? You need a phosphate group on one of the strands to ligate to fragments together. In a ligation with an insert, the phosphate is provided by the insert, but here you only have one fragment (your vector), so dephosphorylating it will make it impossible to self-ligate (which is actually the reason why you do it when you would want to use this vector in a ligation with an insert). So, briefly, just cut with your enzymes of choice, fill in the ends with Klenow and then ligate.


I dephosphorylate to avoid re-ligation with the fragment I cut out (which I have observed previously). After selecting the appropriate band, I fill in the ends with Klenow, and this reaction gives back the retrieved phosphate groups and creates the blunt ends. As I mentioned the ligation control was good, so I don't think that dephosphorylation should be the problem :) Any other idea?

#4 pito

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Posted 22 March 2011 - 11:42 AM

Try an electroporation.

I used chemical competent cells too before and always got bad results.
If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.

#5 dpo

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Posted 22 March 2011 - 02:59 PM

What do you mean with the ligation control and did you separate the bands on a gel?




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