Hello,

How many BglII sites is there between your anchor and the first primer?

You will certainly need to have a good estimation of the background. How many primers do you have?

Germain

Hi Germain,

There are only 2 BglII cut sites between the anchor and the first primer.

And I have one anchor primer and 4 other primers. If the anchor primer is at 0 bp, the other primers are located at -7200, -4000, 4500, 9500.

So far, my PCR reactions have not been optimized properly yet but I see a faint band when using the anchor primer + primer at 4500 bp but not with other combinations.

Jimmy

Jimmy,

Let's make things clear: without cutting and religation, you should have no amplification because all primers are in the same orientation. Am I right? The best thing would be to have at least a negative control between the anchor and the primer that gives a signal. Another useful control would be to have a plasmid or BAC containing the entire locus under study, that you would treat the same way. This would gives you an indication of the reaction efficiency outside a cell, including PCR (see the paper by Hagege et al, Nat Protoc, 2:1722), and confirm that your signal is not due to better priming efficiency of a particular primer pair.

They also suggest to use a 4bp cutter for small loci, as this will reduce background.

Did you consider doing 3c-seq? Although analysis is not simple, it might finally be simpler than 3c-PCR controls!