Reduced emPCR yields

[TonyBrooks]

Hi all
Since the turn of the year we've seen a decline in the yields we're getting from emPCR. Generally we run amplicon Lib-L runs with a very tight amplicon size. The final product is around 480bp +/- 10bp, so we usually estimate concentration based on Qubit and go in with 0.5cpb. Last year, we were getting consistent results of 9-11% enrichment, but recently we've seen that halve, with some runs barely attaining the 2m beads required for the run.
For our last run, we upped the amount of library to 0.8cpb, but we only have a 4.5% enrichment. Has anyone seen anything similar? I can't see anything has changed in our workflow between now and last year.
Cheers
Tony

[RCJK]

Hi Tony,

I have seen similar things recently where my SV titration looked normal, but my LV &/or MV rxns had extremely low yields that did not correspond to the titration cpb inputs. I can get the lot numbers and post them to see if it is something w/ the emPCR kits perhaps.

[TonyBrooks]

We found our problem. Our NaOH was degrading rapidly. Apparently, you store it in plastic, NEVER IN GLASS, in the dark. We've had ours sat in a glass bottle on the shelf in sunlight. Who knew?
A repeated emPCR gave ~13% yield where previously we were getting 4-5%.

[MissDNA]

We were having this problem end of last year. It started when we begun using a new lot of emPCR reagent. I had long discussions with Roche about it. Problem ended when we started using another lot of reagents.

[cliffbeall]

Ha, my thesis advisor used to go on about storing NaOH in plastic. I picked up the habit, but never heard of it making a difference before.

It will also pick up CO2 from the air if you open the solution too much.