4 replies to this topic

[Eureka]

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 ^^

[dpo]

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.

[Eureka]

dpo, on 22 March 2011 - 04:25 AM, said:

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?

[pito]

Try an electroporation.

I used chemical competent cells too before and always got bad results.

[dpo]

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