Help to optain a long PCR produc - (May/03/2012 )

Hi everyone.

I'm working whit mammals cell cultures in which homologous recombination should be occured. In order to check this, I performed two different PCR reactions, one for the 5' end and one for the 3' end. I'll obtain the desired band only in the case that homologous recombination ocurrs.

Due to the low quantity of the starting material I'm not able to check the genomic DNA quality (coming from stem cells).

I obtained the 5' product (around 6 kb) in 5 from the 11 clones that I'm checking. Now I'm trying to set the 3' PCR reaction but until now I have not good results.

I have two different set of primers, and I tried nested PCR (first product 9300 and second product 8000 pb). I'm using the Long PCR Enzyme mix from fermentas.

whith the second pair of primer (direct from DNA) I get some 6 kb and lower products at 57°C, but when I tried whith 58,5 no products were obteined.

I need some advice, what else can I try?

You should be able to get some nice quality genomic DNA from ES cells and even do some Southern blots if you need to. What kind of mouse are you trying to make (knock-out, knock-in, conditional knock-out)?

What are your reaction conditions?

Any particular reason your amplicon needs to be so large? If you are checking for recombination on just one end of the site, a smaller amplicon would be much more likely to succeed, then you can do some sequencing to be absolutely sure, assuming you are making a mouse and would rather not find out afterwards that something went wrong.

If you must do such a large amplicon, check the GC% of the amplicon, if its GC rich, try adding betaine, ethylene glycol, or1,2 propanediol. Easy to read source which cites the original paper: http://bitesizebio.c...-actually-work/
I'll confirm through own experience that these are really helpful, but *only* if its GC rich, otherwise it won't do anything.

Also, what are the TMs of your primers? Matched TMs/GC content of the primers is very helpful in getting clean bands from long range PCR.