Skip to content
New issue

Have a question about this project? Sign up for a free GitHub account to open an issue and contact its maintainers and the community.

By clicking “Sign up for GitHub”, you agree to our terms of service and privacy statement. We’ll occasionally send you account related emails.

Already on GitHub? Sign in to your account

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

Open
wants to merge 14 commits into
base: master
from

Conversation

@teresa-m
Copy link
Contributor

teresa-m commented Nov 7, 2019

No description provided.

Copy link

erxleben left a comment

  • 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_protocols/beer-dna-sequencing.md Outdated Show resolved Hide resolved
_protocols/beer-dna-sequencing.md Outdated Show resolved Hide resolved
_protocols/beer-dna-sequencing.md Outdated Show resolved Hide resolved

- 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)

This comment has been minimized.

Copy link
@erxleben

erxleben Nov 15, 2019

which community? Nanopore? Is that a community knowing about updates?

- 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:

This comment has been minimized.

Copy link
@erxleben
_protocols/beer-dna-sequencing.md Outdated Show resolved Hide resolved
@@ -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

This comment has been minimized.

Copy link
@erxleben

erxleben Nov 15, 2019

Suggested change
- Thermal cycler at 30° C and 80° C
- Thermocycler at 30° C and 80° C
_protocols/beer-dna-sequencing.md Outdated Show resolved Hide resolved
_protocols/beer-dna-sequencing.md Outdated Show resolved Hide resolved
#### 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

This comment has been minimized.

Copy link
@erxleben

erxleben Nov 15, 2019

dont understand this. The wheel of the pipet?

@erxleben

This comment has been minimized.

Copy link

erxleben commented Nov 15, 2019

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

bebatut and others added 5 commits Nov 29, 2019
Co-Authored-By: Anika <erxleben@informatik.uni-freiburg.de>
…nceCommunity.github.io into teresa-m-update_sequencing_protokoll
@bebatut

This comment has been minimized.

Copy link
Collaborator

bebatut commented Nov 29, 2019

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.

Copy link
Collaborator

bebatut left a comment

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

- [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)

This comment has been minimized.

Copy link
@bebatut

bebatut Nov 29, 2019

Collaborator

How much quantity is needed there?


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

This comment has been minimized.

Copy link
@bebatut

bebatut Nov 29, 2019

Collaborator

Which quantity of FB is needed?

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

This comment has been minimized.

Copy link
@bebatut

bebatut Nov 29, 2019

Collaborator

Given before it is written 200, I am not sure to understand how we can not remove more than 30 here


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

This comment has been minimized.

Copy link
@bebatut

bebatut Nov 29, 2019

Collaborator

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


#### Prepare flowcell priming mix
<!--Add a screenshot-->

This comment has been minimized.

Copy link
@bebatut

bebatut Nov 29, 2019

Collaborator

Do you have a screenshot of the software here?

@bebatut

This comment has been minimized.

Copy link
Collaborator

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

Sign up for free to join this conversation on GitHub. Already have an account? Sign in to comment
Projects
None yet
Linked issues

Successfully merging this pull request may close these issues.

None yet

3 participants
You can’t perform that action at this time.