SV Emulsion Breaking

[Dynamac]

I think this topic was addressed in another thread, but I can't find it in the sea of posts. So, I apologize for creating a new thread.

I am about to embark on a 16-sample SV emPCR, and the prospect of breaking 16 separate emulsions with 16 syringes is daunting. Has anyone had success with breaking them by centrifugation?

Thanks.

Barry

[MissDNA]

I don't even know how to break emulsions by centrifugation.
The few times we had to do 16 emulsions in our lab, we actually had enough beads left over from titration to fill up the plate. I think we did 4 titrations a day using 2 points. It is a lot of work though, and I can imagine in a lab where time is money, it would not be feasible to do it.

[ajthomas]

I've done 16 in a day more than once, although when I have that many, I usually try to break it up into a couple of days. I've found a few things that make the process easier and more efficient. You can do the syringe washes two-at-a-time. Draw up the emulsions and wash the wells one at a time, but stop after you screw on the syringe filter. Once you have two, you can do all the washes with a syringe in each hand. You still have to elute from the filter one-at-a-time, though. Use a larger centrifuge, rather than a benchtop mini-centrifuge so that you can spin them all at the same time. It's also easier to rotate the tubes if you can have some space between them. For all the washes, you can use a repeater pipette with a 5ml combitip, rather than the 1ml pipette. Finally, you can usually dump the tubes after centrifugation, rather than draw it off with a pipette. Do all of these things and your breaking and emulsion time and effort will be dramatically reduced.

[MissDNA]

So do you break all the emulsions together, even though they are from differen libraries? We always break emulsions separately and clean up the hood/leave the UV on for 15 min when processing different samples to avoid any kind of contamination by aerosols. This is time consuming as well.

[ajthomas]

Yes, I do them all together. The danger is contamination of future emPCRs, not the current ones. All the DNA you want and what will generate sequence data is attached to the beads, so as long as you keep the beads separate, there won't be any contamination.

The aerosols and what you remove with the melt step after breaking the emulsions contain lots of DNA that is an excellent template for emPCR. Keeping the different parts of the process physically separated (in different rooms/parts of the lab) and breaking the emulsions in the hood are guidelines to prevent those DNA molecules from contaminating future emPCRs.

[MissDNA]

The recommendation Roche gave us was to break different samples separately but what you say makes sense. I guess we are just being extra carefull.

[ajthomas]

That's interesting Roche told you that. Our trainer from Roche didn't say anything like that, and until now I had never heard anyone say they decontaminate the hood between breaking each emPCR reaction.

[MissDNA]

We got our first trainning in 2008 and it was still FLX standard chemistry. I may have been stuck with some misconception since then.
It is good to know is not a necessary step. Surely saves time.