We have the same problem, as we don´t have the Illumina heater specified in the HT protocol, but perform the step in a regular PCR machine. Also because the specified deep well plates in our hands ( without automation) give more variable results than using PCR plates of the same material.
We will use the specified kits and stick to the protocol as close as possible, I think without such exceptions it will be hard to conduct the project.
Do we agree on the compromise ?
Best, Philip
Von: geuvadis_rnaseq-bounces@lists.crg.es [mailto:geuvadis_rnaseq-bounces@lists.crg.es] Im Auftrag von Tuuli Lappalainen
Gesendet: Mittwoch, 5. Oktober 2011 13:26
An: Mathias Brännvall
Cc: geuvadis_rnaseq@lists.crg.es
Betreff: Re: [Geuvadis_rnaseq] Geuvadis mRNAseq kit - further specification
Hi Mathias,
The high-throughput library prep doesn't require robots or automatics, you just need to process the samples on a 96-well plate and have a heat block, a shaker, a centrifuge and a PCR machine for that. Can you check again if following the HT protocol would not be possible?
best regards,
Tuuli
Tuuli Lappalainen, PhD
Department of Genetic Medicine and Development
University of Geneva Medical School
CMU / Rue Michel-Servet 1
1211 Geneva 4
Switzerland
Tel. +41-(0)22-3795550
tuuli.lappalainen@unige.ch
On 10/4/11 12:53 PM, Mathias Brännvall wrote:
Dear Tuuli et al.
Unfortunately, we (Uppsala) can't follow the HT protocol right now since we have not automated all the steps yet. We are supposed to get a robot for library prep soon but it is not absolutely sure it will be in operation in time for these samples. We hope this is acceptable anyway otherwise we would have to wait until we have the robot up and running, which would delay everything even further. The kit will be the same though.
best regards
Mathias
On 2011-10-03 13:19, Tuuli Lappalainen wrote:
Dear all,
I forgot to specify before that we should all use the high-throughput protocol in the library preparation - thanks Peter for pointing this out. No one has had any objections about the kit specified below, so we'll use that in the Geuvadis library preps.
best regards,
Tuuli
Tuuli Lappalainen, PhD
Department of Genetic Medicine and Development
University of Geneva Medical School
CMU / Rue Michel-Servet 1
1211 Geneva 4
Switzerland
Tel. +41-(0)22-3795550
tuuli.lappalainen@unige.ch
-------- Original Message --------
Dear all,
For the mRNA sequencing of the Geuvadis samples, we suggest using the
latest Illumina sample prep kit for HiSeq with 24 indexes:
TruSeq RNA Sample Prep Kit V2 Set A and Set B (RS-122-2001 and RS-122-2002)
As we agreed on the call, you should let me know this week if you don't
agree with using this kit - otherwise we'll go forward with this, and
everyone should to order the kits as necessary to prepare for receiving
the RNAs in the end of October or early November.
Please see the attachment for full information from Illumina for
ordering - as you know, it is absolutely critical that we all follow the
same protocol.
best regards,
Tuuli
--
Tuuli Lappalainen, PhD
Department of Genetic Medicine and Development
University of Geneva Medical School
CMU / Rue Michel-Servet 1
1211 Geneva 4
Switzerland
Tel. +41-(0)22-3795550
tuuli.lappalainen@unige.ch
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--
_____________________________________________
Mathias Brännvall, PhD
Project coordinator
Dept. of Medical Sciences, sect. Molecular Medicine,
Uppsala University
phone:+46-18-6114934
e-mail: Mathias.Brannvall@medsci.uu.se
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