PCR from BAC - PCR from BAC (Oct/23/2009 )

Hello all,
I'm trying to insert a 2kb fragment into a 160kb BAC that had been previously transformed into an E.coli strain of bacteria. I'm using a recombineering method, and, after the insertion process, everything seems pretty good. The experimental plate has good colony growth that selects for antibiotics on both the BAC and the insert fragment while the negative control has very little background. So, judging by that, I believe the insertion process worked (Note* The insert goes through homologous recombination with a region of the BAC, so it should be a site-directed insertion... i.e. the 2kB fragment shouldn't be randomly inserted into the genome or BAC DNA).

However, when I go to verify the insertion worked, I am getting nowhere. I have tried a few methods, but nothing is helping. Method #1 was to use a series of 3 pairs of primers that would amplify the region of insert only if the insert recombined correctly (they would give 1 kb, 1.3 kb, and 2.1 kb fragments in the PCR if correct). The last pair of primers, if the clone insertion did not work, would give a 100bp fragment. Each time I've done the PCR, nothing has showed up. I've even tried to use the original BAC DNA w/o the 2kb insert, but I couldn't see anything. I'm worried that my PCR technique is off. I use a 10ul reaction, but how much BAC template DNA should I be using? Also, should I be using a high processivity Taq like pfu Turbo since the template DNA is so large? The Tm of each primer is >52 degrees.

Method #2 was to do a restriction digest and observe the change in banding pattern, but the issue there is the digerstion creates so many bands that I can't tell if there's a change or not. I'm looking for a better cutter to use, but until then, PCR is the best option.

What to do???

Thank you.

The Tm of your primers sounds quite low. How many bases are they? I would probably prepare BAC DNA and send it for sequencing, which will tell you more about exactly what is happening. Meanwhile, work on getting the 100 bp fragment pcr working on your parent BAC. If you get that working, then the same primerrs can be used to detect the insertion, although this is not quite as good as having a primer on the insert and a primer on the BAC. You can also debug your primers on the insert-only DNA fragment, amplifying up the insert.

You might want to think about the possibility that you have cells with the BAC and with the insert plasmid, but no recombination. How are you selecting against this situation.