emPCR issues

[jOn]

Enrichment following SV emPCR has quite dramatically increased with the last few preps. We're well used to doing a range of library preps and subsequent emPCR for both amplicon and shotgun using the SV kit and have up until now had few problems. Has anyone else out there had emPCR issues lately - if so have you had any feedback from Roche? Reading the other posts on here it seems to be a recurring theme at the moment.

[Soulbee]

Hi, Im carrying out MV emPCR, (oil: 93777520; kit: 93786520) and a majority of my emulsion has come out broken. I have not had any problems in the past with broken emulsions and these are libraries that have been successfully sequenced in the past (~couple of months ago) and nothing has been changed except for using of this kit. Roche inquired about the thermocycler lid temp and tissue Lyser but they have been working fine in the past and still are now. I am waiting to hear back from Roche.

[RCJK]

Hi, a few months ago I had a streak of broken emulsions across different lots of MV and LV oils. It ended up being the seals I was using to seal the plates with. I was skeptical that this was really the cause, but did some side by side reactions with plate seals and strip caps and the strip caps were always fine while the seals had many broken emulsions. They were the same plate seals I've always used, but perhaps they were a different lot or package of seals. They were ABI plate seals by the way. Don't know if this helps, but it's worth looking into. I was even seeing emulsions break prior to thermocycling w/ the seals. They suspected the tissue lyser and everything else too.

[Soulbee]

Thanks for the input, we are going to also try using a different sealing method because the new oil formulations may be more sensitive to heat, which may be why im seeing lots of broken emulsions.

[jOn]

Guys I've been using plastic strip caps and the usual low bind plates from eppendorf (always have) and never once had a broken emulsion - my problems appear to be new oil related.

[Soulbee]

I tested different lids (strip caps, adhesive and two different kinds of heat seals) - The heat seals gave the best results and the adhesive one resulted in lots of broken wells. The new oil is definately variable but with LV, i haven't had much of a problem. I have been having problems with MV oils and I cannot tell whether it is the reagents or the MV oil that is giving me broken emulsions.

[pmiguel]

With the new oil formulation, is it not normal to see clear oil at the tops of emulsions after thermal cycling now? If so, how does one detect a "broken emulsion"?

--
Phillip

[Soulbee]

With the new LV oil, i have noticed a distinct clear layer at the top, but I have only used a couple of these oils.

Broken emulsions can be distinguished from the non-broken ones by looking at the bottom of the emulsion wells. I usually hold mine up at an angle at a good light, and if an emulsion is broken, there is a pellet of white capture beads at the bottom of the wells. I imagine as microreactors break, the once-contained capture beads settle to the bottom. Sometimes it helps to open the lid and hold it up to the light, and look at the bottom of your plates from under. Sometimes it can be very hard to see because the round bottom of the wells may cast a shadow making it seem as if there is a pellet while there is actually not. Anyway, looking for settled DNA beads at the bottom i think is a very good way to tell if an emulsion is broken and has been working for us so far.

[EJS]

MV-emPCR

My titration run (sv-emPCR) had very nice enrichment %. However, with same cpb that worked well for sv-emPCR, mv-emPCR enrichment % came out to be ~ 30 to 50%. I have repeated the experiment with different lot oil but ended up with same bad result. The lot # for MV-emPCR oil are as followed:
93805620 and 93815320.

[pmiguel]

I think we have seen discrepancies between SV and other scale emPCRs. But 30-50% is really high. Did you try repeating the SV? Possible there was a 10x error in a calculation somewhere?

--
Phillip

[Soulbee]

I dont think so, because two of the four samples were repeats, and have worked before in the past with LV emulsions.. We are going to try running the samples with LV oils instead. Thanks!

[jOn]

It is the oil

We've had 50% enrichment with the new SV oils even after halving the cpb. Switched back to a stock of old oils last week and the problem disappeared. There is definitely something different about the new oil we’ve been using. Drop me a message, I’ll be happy to share lot numbers. Roche have told me to forget what I know about titrations from previous experience and start again…………..so much for the yellow note that accompanied the new oils. Soulbee – your problem sounds identical to mine – get on to Roche, they should be well aware of these issues by now.

[EJS]

SV titration with new oil

I've repeated my sv emPCR with new oil (my previous sv emPCR was using old oil but not expired...) and in order to reach ~10% enrichment, I had to use ~70 to 90% fewer library compare to old oil. My new ideal cpb is from 0.25 to 0.5 compare to 4 to 8 with old oil. I am still trying to troubleshoot some other issues. I will keep you updated...

[pmiguel]

How are you determining the "c" part of "cpb"? Using the dye on rapid adaptors, or some other method?

--
Phillip

[crndy52]

I too am having problems with my emulsions. I get the recovery rates around 7 to 8 % doing SV titrations only to find that when I do the LV emulsion my recovery rate is twice as much or more. The emulsions are usable but the Dot/Mix percentage goes up with the recovery rate and the data comes down. I have been in contact with Roche for more than a month on this issue but have not gotten anything from them thus far. Has anyone gotten any usable information from Roche on this issue?