SBI Minicircle DNA Kit -- No colonies -- ligation / transformation problem - (Jul/22/2013 )
I am using SBI's Minicircle DNA kit (http://www.systembio.com/minicircle-dna-vectors/overview). The idea is that you create a parental plasmid, in my case consisting of their vector + my shRNA, and I cannot get it to work. I linearize the vector (MNSI506A-1) using EcoRI and BamHI (my insert has the correct ends for a sticky ligation). After linearization that is verified on a gel, I attempt to ligate in my insert (stem-loop structure), and then transform the ligated plasmid into E.Coli. After transformation, I plate on LB + Kan plates, but I see no colonies. I think the error occurs when I create my insert (anneal two oligos to create the stem-loop) or during the ligation. The backbone is ~7500bp and the insert is 60bp. I have allowed the annealed oligos to cool slowly, and have done my ligation overnight at room temp. The concentration of my backbone is ~30 ng/uL after being gel purified, and the insert is ~100 ng/uL. I have also tried to transform various amounts of DNA into the E.Coli.
If you read all that, a million times thank you. Any suggestions as to where this is going wrong would be greatly appreciated.
If you are ligating oligos, the 5' end needs a phosphate, either from the synthesis or a PNK treatment.
When I called the company and asked about this specifically, they told me that there should NOT be any 5' phosphate. I find it hard to believe they are incorrect, but perhaps I should order one set of oligos with the phosphate and see what happens. Thoughts?
Also, I am ligating an oligo to a digested vector, not two oligos to one another.
Well, given that your vector still has 5' phosphates, you shouldn't in principle need the oligos to be phosphorylated, although you'll create a nicked product. More worrisome is that you get zero colonies. I would assume your RE cutting is not 100% efficient, and it is very likely that you should be getting some parent plasmids transforming. Your competent cells may be no good. A control transformation with 10 pg of pUC19 would tell the story.
Also, it is very likely that you have 10000x too much oligo in your ligation mix. You want about equimolar amounts, which means you must dilute your oligo 100x to 10000x. If you don't, then you ligate two different oligos to each end of a vector, and nothing further happens.
When I performed the transformation, I included a cut vector (negative control) and an uncut vector (positive control). The positive control had a lawn of colonies showing that the transformation is not where the error is occurring. I diluted the oligos to 20 uM as instructed in the protocol, and had about 3:1 ratio of insert:vector so that the insert isn't limiting.
I should say that after plating a majority of the transformation and seeing now colonies, I took the remaining transformation, spun it down, and re-suspended in less media. This yielded a few colonies on about half the plates. I did not feel confident with the result, but perhaps there is just very poor efficiency due to the nicked product.
The lawn unfortunately does NOT tell you the competent cells are good enough for cloning. You need high competence, not just some competence. You need to calculate a transformation efficiency, in CFU per microgram of DNA. It should be 10^8 or higher.
Diluting your oligos to 20 uM is ok, but you need to dilute a LOT more. The 1:3 ratio is a ratio of MOLES not of volume. There are many more moles of oligos in a microliter than there are moles of your vector, if they have the same DNA concentration. About 120 in this case (7500/60).
Good point regarding the competency of the cells. In terms of the oligos. The 20 uM concentration is for the annealing reaction. I dilute further using nanodrop to ensure the correct ratio of vector to insert.