SEQanswers

Go Back   SEQanswers > Sequencing Technologies/Companies > 454 Pyrosequencing



Similar Threads
Thread Thread Starter Forum Replies Last Post
QR Codes and Plate Maps rfrichholt Bioinformatics 1 12-21-2011 03:46 PM
Pool amplicons on a plate tng012 454 Pyrosequencing 3 07-09-2010 11:52 PM
Agencourt SPRI Bead Plate squeak General 0 07-01-2010 10:21 AM
Cost Illuminia GIIx sequencing per plate? MicroOmicsMan Illumina/Solexa 1 06-11-2010 02:19 AM
step by step for rarefaction calculation psong Metagenomics 1 01-06-2010 05:08 AM

Reply
 
Thread Tools
Old 12-02-2011, 07:37 AM   #1
HMorrison
Senior Member
 
Location: Massachusetts

Join Date: May 2009
Posts: 116
Default Plate sealers for emPCR step

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.
HMorrison is offline   Reply With Quote
Old 12-02-2011, 08:17 AM   #2
Nicole 454 Sequencing
Member
 
Location: Branford

Join Date: Apr 2011
Posts: 19
Default

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

Last edited by Nicole 454 Sequencing; 12-02-2011 at 08:23 AM.
Nicole 454 Sequencing is offline   Reply With Quote
Old 12-02-2011, 08:19 AM   #3
HMorrison
Senior Member
 
Location: Massachusetts

Join Date: May 2009
Posts: 116
Default

Okay, okay...just trying to make life easier for the RAs.
HMorrison is offline   Reply With Quote
Old 12-02-2011, 10:33 AM   #4
pmiguel
Senior Member
 
Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
Posts: 2,317
Default

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
pmiguel is offline   Reply With Quote
Old 12-02-2011, 01:02 PM   #5
MissDNA
Senior Member
 
Location: Brazil

Join Date: Nov 2010
Posts: 146
Default

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 donīt 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...
MissDNA is offline   Reply With Quote
Old 12-06-2011, 07:39 AM   #6
Nicole 454 Sequencing
Member
 
Location: Branford

Join Date: Apr 2011
Posts: 19
Default

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
Nicole 454 Sequencing is offline   Reply With Quote
Old 12-06-2011, 08:06 AM   #7
MissDNA
Senior Member
 
Location: Brazil

Join Date: Nov 2010
Posts: 146
Default

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.
MissDNA is offline   Reply With Quote
Old 12-12-2011, 04:36 AM   #8
yaximik
Senior Member
 
Location: Oregon

Join Date: Apr 2011
Posts: 205
Default 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...
yaximik is offline   Reply With Quote
Old 12-21-2011, 11:11 AM   #9
Indiana
Junior Member
 
Location: Indiana

Join Date: Nov 2011
Posts: 5
Default 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.
Indiana is offline   Reply With Quote
Reply

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 10:43 PM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO