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added discussed changes of the last sequencing round to the protokoll #60

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Merged
merged 14 commits into from
Dec 19, 2020
Merged

added discussed changes of the last sequencing round to the protokoll #60

merged 14 commits into from
Dec 19, 2020

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teresa-m
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@teresa-m teresa-m commented Nov 7, 2019

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erxleben
erxleben suggested changes Nov 15, 2019
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  • line 77: Store the library on ice until ready to load onto the flow cell
    add: The mix you have prepared is called sequencing library or just library
  • line 122: which size

_protocols/beer-dna-sequencing.md Outdated

- Please search for "MinION Software" in the Nanopore downloads web page https://community.nanoporetech.com/downloads.
- Select your host operating system and follow the link to the installation instructions.
- Since the supported systems and the installation procedure is constantly updated, please always refer to the community website for details. (You need to have an account to access the community website)
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which community? Nanopore? Is that a community knowing about updates?

_protocols/beer-dna-sequencing.md Outdated
- Select your host operating system and follow the link to the installation instructions.
- Since the supported systems and the installation procedure is constantly updated, please always refer to the community website for details. (You need to have an account to access the community website)

### Setting up the software:
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delete

_protocols/beer-dna-sequencing.md Outdated
@@ -28,18 +55,19 @@ The most important: **7.5µl DNA** extracted as described in the [DNA extraction
- Fragmentation Mix (FRA)
- Rapid Adapter (RAP)
- Nuclease-free water (e.g. ThermoFisher, cat #AM9937)
- 0.2 ml thin-walled PCR tubes
- 0.2 ml PCR tubes
- Ice

##### Needed material
- Thermal cycler at 30° C and 80° C
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Suggested change
- Thermal cycler at 30° C and 80° C
- Thermocycler at 30° C and 80° C

_protocols/beer-dna-sequencing.md Outdated
#### Prepare loading port
1. Open priming port to check for small bubbles
2. To remove bubbles take some liquid from the port by use the P1000 pipette set to 200 µl
3. Remove the liquid by turning the weel but only until 220-230 µl
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dont understand this. The wheel of the pipet?

@erxleben
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very first round done. But needs more improvement. Will work on it again

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bebatut commented Nov 29, 2019 •
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I did some rearrangement of the content.
I moved the requirements at the beginning to help people to prepare everything before starting to follow the protocol. Let me know if these changes are ok for you, otherwise I can revert them.

I also did some formatting to support the PDFs export in different version.

bebatut
bebatut reviewed Nov 29, 2019
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Just some comments/questions on the content. We should also add some images, specially of the flowcell with the different ports

_protocols/beer-dna-sequencing.md Outdated
- [Rapid Sequencing Kit](https://store.nanoporetech.com/sample-prep/rapid-sequencing-kit.html) including
- 30 µl Flush Tether (FLT)
- 34 μl Sequencing Buffer (SQB)
- µl Flush Buffer (FB)
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How much quantity is needed there?

_protocols/beer-dna-sequencing.md Outdated

Either you find the "MinION Software" botton directly or you open it via the terminal using the path "/opt/ui/MinKNOW"
3. Prepare the **priming mix**
1. Add 30 µl of Flush Tether (FLT) directly to the Flush Buffer (FB) Eppi tube
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Which quantity of FB is needed?

_protocols/beer-dna-sequencing.md Outdated
Requirements:
- You have to have a account at nanopor MinION to start the software
- The divice need to be attached to the computer to start the software
Note: removing more than 30 µl will damage the pores in the array because they need to be covered by the buffer at all times
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Given before it is written 200, I am not sure to understand how we can not remove more than 30 here

_protocols/beer-dna-sequencing.md Outdated

5. Gently replace the SpotON sample port cover
6. Making sure the bung enters the SpotON port
7. Close the priming port and the MinION lid
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Will the sequencing start automatically after? What happen in the software?
We should probably add also a screenshot of the software but also way to check that everything is going well

_protocols/beer-dna-sequencing.md Outdated

#### Prepare flowcell priming mix
<!--Add a screenshot-->
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Do you have a screenshot of the software here?

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bebatut commented Jan 21, 2020

I did quite some changes given the last discussions:

  • Duplicate requirements to have them at the beginning of the protocol but also when needed
  • Customize content for minion or flongle flowcell
  • Expand the steps with details (given the content on the Nanopore website and external resources) and schemas

I think this protocol is now ready for review.

The different PDF can be downloaded there for review: https://send.firefox.com/download/2ca94dfb1552e9f5/#sZoUQCifeM4wGFhjscSWYw

@mmiladi mmiladi mentioned this pull request Sep 29, 2020
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@bebatut bebatut merged commit 9f00d96 into StreetScienceCommunity:master Dec 19, 2020
@bebatut bebatut deleted the update_sequencing_protokoll branch December 19, 2020 14:10
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@teresa-m @erxleben @bebatut