Hello Caitlin,

Overall it seems the PCR conditions for your floxed gene is suboptimal. Although the PCR works well for the cre, it may be related to the quality of the DNA. But since DNA quality is good enough to amplify the cre, the best way would be to tweak your PCR protocol.

1- ,If you are not using such a protocol already, I would suggest to use Touchdown PCR protocol. We use that and it works like charm. The only difference with a regular PCR protocol is that you set the annealing temperature to 65°C and then decrease the annealing temperature by 0.5°C every cycle over 30 cycles.

2- If above does not work and if you have the sequence of the floxed gene, design new primers. Ideally, you would like to have some primers to amplify both the floxed and wt allele or a primer pair that gives 2 products of different length so tha you have a kind of dual validation.

Hope this helps

Nick

Hi Caitlin,

I would agree with Nicolas, the PCR for your floxed gene is probably weaker then for the Cre, it happens often. In addition to what Nicolas suggested, I would bring forward two more points:

1. Don't underestimate the DNA quality. If a set of primers works well on a certain DNA it does not mean that the DNA quality is optimal, it means that for that set of primer the DNA quality is fine. I don't know how you extract your genomic DNA, but of course there are several possibilities: PK digestion, boiling and spin down; PK digestion, saturated NaCl extraction and EtOH precipitation; PK digestion, Phenol/CHCl3 extraction and EtOH precipitation; PK digestion and columns; Kits with milder proteases, boiling and spin down... etc. There are PCRs that require the best DNA purity to be reliable. So if your DNA is prepared without any extraction step, I suggest for this specific PCR to add at leas a saturated salt extraction step.

2. You can push the PCR reaction in different ways, besides the step-down approach. For example primer concentration can go up 1 uM on genomic DNA (most PCRs will work with 0.25-0.5 uM....). You can increase the amount of Taq polymerase, mostly if you are using a cheap enzyme for genotyping purposes. For specific PCR reactions 1 u might not be sufficient to guarantee reliability, better use 2 u. You can increase the annealing time: there are primers with slow binding kinetics and a 15-30 s annealing time might be too short, try 60-75 s, it will take a bit longer for the PCR to complete, but you might achieve reliability.

Good luck!