[Anthony.287]

Usually I do titrations with SV, but this particular client and particular project wanted a tradeoff between SV titration and number of reads, so I've been doing small-range titrations with the MV kits.

Those ratios take into consideration the volume of all of the liquid added to the volume of the oil.
SV - 513-522ul aqueous : 600ul oil
MV - 1,896-1,915ul aqueous : 2,000ul oil
LV - 18,851-18,950ul aqueous : 10,000ul oil

The volumes for aqueous are given in a range because of the ranges given in the protocols (1-10, 1-20, 1-100) for volumes of DNA to add. They also take into consideration the volume of the capture beads added. These volumes were given to me by an FAS, but they don't seem to match up with what I've experienced.

[Rachael]

Hi Anthony,

That's great news that your emulsions are all working again with the MV's.

The tube on the left is the normal enrichment bead tube that comes in the emPCR breaking kit. The tube on the right was the same reagent, however it had a lower density of beads which caused us problems with our emulsions.

Rachael - thanks for the picture! One question; which tube is which?

After contacting Roche, I figured out that the reagents weren't the problem. The problem was that I was using the LV kit, when all of the titrations had been done with the MV. Apparently, the ratio of aqueous : oil is very important when creating the emulsions, and the ratios aren't maintained across the 3 different sized kits. For SV, the ratio is ~0.85-0.87, MV ~0.94-0.96 and LV ~0.88-0.90. I had assumed that the ratios would be the same, or at least very close to the same, across all of the kits, but it sure doesn’t look like it. Please feel free to double-check my math, but I think it’s right.

I went back to using the MV kit with the previously determined ideal cpb, and all is well.

[MissDNA]

Roche is going to replace all the boxes we have in stock from lots 93848420 and 93852420.

Thanks again, Rachel, for telling us about this problem.