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Old 07-08-2015, 12:04 PM   #1
Loren Joseph
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Location: Cambridge, MA

Join Date: Dec 2014
Posts: 2
Default MiSeq prep ancillary equipment

We have ordered a MiSeq. I have several questions about ancillary equipment. Saw only one partially related post.

37 degree forced air incubator”. A list of additional recommended equipment calls for such an incubator. The only place where this seems to apply in a protocol (like the TruSight Tumor Sample Prep) is for the extension-ligation step. The only forced air incubators I find and those which the company recommends are essentially 3 foot tall tissue culture incubators. Unfortunately bench space is sorely limited. My first thought was ‘how about a heat block (with a lid)?’ but a 2nd mention in the protocol specifies “not a heat block” . Drat. Apparently the microplate – filter assembly is too large for heat blocks. Can anyone recommend alternative devices? Alternative protocols?
Library prep in microtubes rather than microplates. The protocol recommends running 16 preps at a time. Some of the steps in the standard protocol, such as using filter plates, would have to be substantially modified, but there are certainly suitable alternatives. Has anyone tried this or do you just use a few wells on the plate?
Centrifugation of microplates at 20 deg. I was told not to cavalierly interpret this as ‘roughly room temp’. The protocols I looked at call for spins up to 10 minutes and forces of up to 2,500 g. The inexpensive plate spinners I was planning on for ‘pulse spins’ do not have timers and do not have temp control. Any recommendations?

Other protocols for cancer sample, such as Qiagen’s “Reader” panels or Archer’s panels seem to have fewer requirements but perhaps they have been less extensively vetted.

As you can tell I am a newbie to NGS, all advice welcome.
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Old 07-14-2015, 11:44 AM   #2
microgirl123
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Location: New England

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What kind of library preps are you planning on doing? The MiSeq itself doesn't require any other equipment except a heat block or thermal cycler for heat denaturation of certain types of libraries.

I certainly wouldn't rush out and buy all the equipment for all the different types of library preps!
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Old 07-15-2015, 03:40 AM   #3
Loren Joseph
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Location: Cambridge, MA

Join Date: Dec 2014
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Default Thanks re MiSeq answer

Microgirl123 - this reminds me that I should have registered with a nom de plume.

Thanks for confirmation of my suspicion/concern. Several companies supplying assays targeting nearly the same oncogene/suppressor panel all use the MiSeq sequencing portion (of course) but with seemingly more robust lib prep protocols. I had hoped to use the Illumina panels, thinking they are the most widely used. The Illumina panels might be more flexible than tech support can say, but for now I will go with another vendor for the library prep.

Regards
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Old 07-16-2015, 10:58 AM   #4
Angelmass
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It is still unclear what types of preps you are doing, but I will say I'm a bit of a newbie in terms of background knowledge, but average about 6 MiSeq runs/week. The only other equipment that we use are Qubit/Tapestation for quant and library size calculations, thermocyclers for PCR reactions, and we denature with NaOH rather than heat.
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