Hello
I have been working TOPO TA Cloning kit of Invitrogen. I was trying to clone a fragment of 1,2 kbp obtained by PCR inside the vector (pCR 2.1-TOPO 3,9 Kbp) of the kit.

In my first tentative I used the PCR product purificated with AC. Amonium and isopropanol. Growing of colonies goes ok. I checked colonies by direct PCR from the colonies with primers of the plasmid on sides of the cloning site and obtained only a 15-20% of fragment of the expected length (1,3-1,4 kbp). In most of the other i observed that the plasmid took inside small fragment, probably primer dimers, and in some of them a fragment of 1,9 kbp.

Then I decide improve my purification. I "re-purificate" samples (previously puricated with Ac. Am-Isop.). Some of them with agarose gel puricatión (quiagen kit) and others with column purificatión (also quiagen kit). Then I checked in a gel and also with spectophotometer the presence of DNA and righth bands in my samples. And with this a repeat the cloning process.

Colonies growing was ok and I cheked by PCR (12 clones x 16 samples: 192 clones) and I obtained 95% of bands of 1,9 kbp!!!. Really nice bands with an straing length. This happenned in both kind of samples, gel purificatíon and column purification ones. Only one of the clones has the rigth length (1,3-1,4).

I have no idea what is the 1,9 kbp fragment that is inside the plasmid?

Could it be a contamination? In the first tentative this fragment appear, but with really low proportion. Could it be an artifat? Could i have any posibility of finding my fragment (1,2 kbp) inside the strain long one(1,9kbp)??

Anyone can give me some advise? thank you in advance!!!

Carmelo Andújar

Hi,

First of all: I don't believe in colony PCR... why? let me tell you what happened with me:

I was trying to check if colonies have my recombinant plasmid and I was doing it by PCR. I got a fine band with the size of my gene... when I digest miniprep with restriciton enzymes I discovered that vector was empty... "but, but, PCR was positive..."
Some days latter (after check all of other possible contaminations) I realize that i was amplify bacterial DNA and a fragment with the same size of my gene... Yes, hard to believe but true. Proved by a control with empty DH5a.

So did you make a control using empty bacteria?

I don't know if this is what happening in your case but...

Good luck

BadKarma