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Old 10-27-2016, 12:57 AM   #1
glovesfox
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Default Other lab equipment for library prep???

Hello everyone,
I'm trying to gauge what equipment we will need for Nanopore library prep for both amplicons and shotgun sequencing. If anybody could post an equipment list that would be really useful please. For other (454) seq a bioanalyzer and Qubit have been fairly essential - does the same apply to Nanopore?

Thanks
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Old 10-30-2016, 04:07 AM   #2
gringer
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If you want to talk about absolute essentials for Nanopore, then the following are needed for sample prep for the 15min rapid transposase kit:
  • Pipettes + tips (P200, P10, P2)
  • DNA sample source containing free DNA
  • Clear Water
  • Minicentrifuge, or fast windmill hands
  • Warm hands (30C)
  • Hot liquid (70C, e.g. coffee)
  • TA Ligase + buffer (0.2l per sample)
  • Small sample tubes (e.g. 1.5l)
  • ONT Rapid Sequencing Kit

The following are nice to have (but not necessary) for the transposase kit (in addition to above):
  • Pipettes + tips (P1000)
  • Nuclease-free water
  • Heating block (70C)
  • Fluorescence-based quantification device (e.g. Quantus, Qubit)
  • Magnetic RNA/DNA purification beads + rack (e.g. Ampure XP)

The bioanalyzer is a somewhat pointless. If you're doing the sequencing anyway, then you get the sample fragment length distribution by looking at the read length distribution after about 10-30 minutes.

ONT is planning to bring out a 5min kit by the end of the year that does away with the need for the ligase buffer and 70C heating step.

Last edited by gringer; 10-30-2016 at 04:13 AM.
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Old 10-31-2016, 12:16 AM   #3
glovesfox
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[*]Minicentrifuge, or fast windmill hands[*]Warm hands (30C)[*]Hot liquid (70C, e.g. coffee)


Ha, I love it. Thanks for the reply.

Do you know if I can get hold of the protocols anywhere prior to buying the kit? I can't seem to find them anywhere obvious on Google and don't have access to the Nanopore Community (where I assume they are available).
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Old 10-31-2016, 12:26 AM   #4
gringer
David Eccles (gringer)
 
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I'd suggest emailing Oxford Nanopore directly about that (support@nanoporetech.com). The protocols change frequently, and there are quite a lot of different kits.

If you're just interested in the general idea about the protocols (rather than specifics) and/or required equipment, then that information is available in the nanopore store. For example, here's the current public information about the rapid sequencing kit. This includes a schematic of how the sample preparation is carried out:
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