Plate sealers for emPCR step

[HMorrison]

Anyone use plate sealers instead of strip caps for emPCR? If so, what type(s) work well? Have used MicroAmp strip caps all along based on 2006 training even though we're using a cheaper 96 well plate.

[Nicole 454 Sequencing]

Hi HMorrison

I stongly recommend sticking to the strip caps. It has been shown that plate sealers do not create as good of a seal during thermocycling. We have seen this at many customer sites, with side-by-side experiments done showing this performance difference. Some consequences to using plate sealers may be:

-Broken Emulsions
-Inconsistent enrichments
-Lack of correlation of SV titration to MV/LV
-High enrichment
-High % Mixed

If you are looking for other plates or strip caps which work well we recommend:
96-well plates, semi skirted Fisher Scientific E951020303
Cap strips for 96-well plates Fisher Scientific E0030127498

These and other recommended materials can be found in the customer accessible area of our website: (http://my454.com/).

I hope this helps!

Nicole
Technical Support Scientist
454 Life Sciences, A Roche Company

[HMorrison]

Okay, okay...just trying to make life easier for the RAs.

[pmiguel]

Just as a caveat: we had been plagued by inconsistent enrichments even though we were using strip caps. Finally noticed that we were using strip caps made by a different company to the plates we were using!

We went to the right kind of caps and results have been much more consistent since.

--
Phillip

[MissDNA]

We have always used sealers. That was what we were taught in our training in 2008, and we stuck with it.

We never seen broken emulsions, but we do observe lack of correlation of SV titration ro MV/LV and inconsistent enrichment. No Roche representative has ever suggested we should change to strip caps.

Yesterday we did a full LV, same sample in both cups: in one cup we got a bit over 2 million beads and in the other a bit over 4 million. Our SV titration was 8% enrichment at 16 cpb, we chose 13 cpb because we know things don´t correlate well.

I wonder if we switch to caps our results will get more consistent.

Another lab here in Brazil is having a terrible time with enrichment, and they started counting the amount of beads/tube and they hardly ever find 35 million beads as stated in the manual. In that case is really hard to find correlation...

[Nicole 454 Sequencing]

Hi MissDNA,

Do you know what kind of plates, caps or method of aliquotting the other lab is using?

I would be extremely interested to see the results of a side-by-side comparison of strip caps vs. plate sealers done in your lab. In my experience at customer sites this has improved the correlation of the SV titration to MV/LV emulsions and also improved consistency between within platforms.

Another finding (as Suefo brings up in an earlier post) was the use of the Matrix pipettor for aliquotting having a similar effect. The matrix pipettor and plate sealers are an artifact of our older technology and we have since made many changes to our product.

These changes may not work well with older recommendations, but our most recent recommendations can be found in the link provided above.

Nicole

Technical Support Scientist
454 Life Sciences, A Roche Company

[MissDNA]

Hi Nicole,

I am not sure what they use to cover their plates, but I know they use plates from Applied in their Veriti termocyclers. We use Veritis here too, and we were told before by Roche people that some problems had been observed when labs used Veritis. We do not use Applied plates.

We have tons of sealers here but we could try at some point do the comparasion.

We sequenced yesterday the beads I mentioned on my previous post. We used 2 million beads per region, and in one region we put the beads with higher enrichment and in the other the lower. The difference was remarkable. Region 1 (higher enrich. beads) had 56,06% passed filter wells, being that 24,82% failed on dot+mixed (mostly mixed) whereas Region 2 (lower enrich. beads) had 71,54% passed filter wells and only 8,82% failed dot+mixed. We had sequenced before beads with different rates of enrichment in different regions but we never observed such variation. It is really weird considering it was the same library, same kit and same person preping the samples.

Thanks for your input.

[yaximik]

Plate sealers

I am using plate sealers, those from Invitrogen for real-time PCR work fine. A few failed occasions indicated that sealers require a good rigid plate, like Eppendorf twin-tech; some flimsy brands like from Fisher and even from ABI tend to fail. But I guess caps should certainly seal more reproducibly, it's hard to argue against this...

[Indiana]

Plate sealer works great

Our setup never fails us. We use:

1) USA Scientific TempPlate Semiskirt 96-well plates (1402-9200)

2) ABGene Thermosealer (AB-0384/110)

3) Thermo Peelable Heat-sealing foil (AB-0745)

4) Eppendorf Mastercycler with silicone pad on top of sealed plate.