inverse PCR (help me please...T_T)

[rarhong]

Does anybody doing inverse PCR here??
I'm not sure about the concentration to make the digested DNA circular. I read in a journal that the DNA concentration at ligation reaction must be low in order to enchance self circularization. I've tried the concentration ranging from 0,1-1 microgram/ml. But I'm afraid that small amount of DNA gone after extraction with phenol:chloroform followed by precipitation. Does anybody know any idea how to keep does DNA from missing?? My PCR always failed.
How can we know that the DNA has been circularized?? The low concentration made it difficult to see it through agarose gel.

Big big thanks for anybody who want to help....

[SebaQ]

Are you using Phenol:Chloroform to purify the DNA from the digestion and ligation steps?

If so, I recommend you to use a better purification procedure, like this one I shared:

[Only registered users see links. ]

or use kits like wizard kits from promega.

Phenol:Chloroform is useful when isolating DNA from bacteria, because you have soooo many proteins to get rid of, and in much more quantities than your ligation or digestions steps. Futhermore, as bacteria has many plasmid copies and you normally process millions of bacteria, the multi-stepped phenol:chloroform isolation procedure works fine, but to keep safe low DNA concentrations, you need a protocol with less steps, for the more steps you have, the more DNA you lose between steps.

How can you know DNA has been circularized? Well, I think you can do this (you will need more concentrated DNA, so first you need to achieve that):

Take a fraction of your theorically circularized DNA and separate it in two tubes. Digest lightly one of the tubes with a restriction enzyme you do know it cuts only once in your known sequence. Let's suppose it doesn't cut anywhere else in the unknown sequence. Don't digest the other tube.

So now load in a gel both tubes: the digested one and the undigested one.

If DNA was recircularized, you will have a light "shift" in the undigested reaction compared with the digested one, because circular DNA migrates diferentially than linear DNA.

If DNA was not recircularized, in the undigested reaction you will have a band, BUT in the digested reaction you will now have 2 bands, because the linear DNA was digested in some point in your known sequence.

Hope it helps ^^!