eamag/protocols-with-steps huggingface dataset:
TITLE: Protocol 1: PCR
AUTHORS:  

[STEPS]
?. Find a Sequence You will need to look up the gene for the sequence you are trying to amplify and download your file (any type described above)
?. Go to PrimerQuest ToolYou will need to go to the PrimerQuest Tool webpage, https://www.idtdna.com/Primerquest/Home/Index, in order to insert file type of choice
?. Select a primer
?. Take a screenshot of potential primers found on IDT then upload them to your lab notebook
?. Confirm your primer. The confirmation page will compile all of the necessary primer information onto one page so that you can have one last check of the aspects of your primers before you order. When you are confident in your choice, save the primer information and send them out to be synthesized.
TITLE: Visium Direct Mount FFPE v3 -- University of Minnesota TMCs
AUTHORS: Laura J Niedernhofer, David A Bernlohr

[DESCRIPTION]
The Visium Spatial Gene Expression for FFPE is designed to measure mRNA in tissue sections derived
from formalin fixed & paraffin embedded (FFPE) tissue samples and requires a Visium Spatial slide
with intact tissue sections as input. 

Tissue sections are processed with Visium Spatial Gene Expression assay performed by the University of Minnesota Imaging Center and sequenced by the University of Minnesota Genomics Center.



[STEPS]
SECTION: Deparaffinization, H&amp;E Staining, Imaging &amp; Decrosslinking
1.   

SECTION: Library Preparation &amp; Sequencing
2.   
 



SECTION: FASTQ Generation
3. BCL data from Illumina sequencer is demultiplexed and converted into FASTQ format using bcl2fastq software version 2.20.0
TITLE: PCLS Viability Assay Protocol
AUTHORS: Morrisey Lab

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">PCLS Viability Assay Protocol- Calcein AM/ethidium homodimer-1 (“LIVE⁄DEAD®) staining and quantitative image analysis</span><span style = "font-weight:bold;font-style:italic;">(</span><span style = "font-style:italic;">Thermo Cat # L3224</span><span>)</span></div></div>

[STEPS]
TITLE: Egg Prep   for Bleach Synchronization (Cabreiro Lab)
AUTHORS: Saul Moore

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Egg prep protocol for bleach synchronisation of </span><span style = "font-style:italic;">C. elegans. </span></div></div>

[STEPS]
?. [Prepare worms]
Leave the Falcon tube to stand for a while until the worm settle to the bottom in a loose pellet.
?. [Egg Prep - Bleaching]
Add 350μL bleach mix to the 2mL solution of worms in M9
?. [Prepare worms]
Wash worms off the plates with a few mL of M9 buffer into a 15mL Falcon tube.
?. [Prepare Bleach Mix]
Mix together in an Eppendorf tube:
[NaOH [4M]]
[Sodium Hypochlorite [Acros Organics, 10/15% active chlorine, 7681-52-9]]
?. [Prepare worms]
Remove the supernatant leaving 2mL M9 solution in the tube (with the worms pelleted)
?. [Egg Prep - Bleaching]
Vortex for 5 min (checking every 30 seconds under a microscope to see if the worms are broken apart and eggs have been released).
?. [Egg Prep - Bleaching]
Once the majority of eggs have been released, quench the solution by topping up the Falcon tube to 14mL with M9.
?. [Egg Prep - Bleaching]
Centrifuge at 6700 rpm for 2 minutes to pellet the eggs to the bottom.
?. [Egg Prep - Bleaching]
Carefully remove the supernatant using a plastic Pasteur pipette.
?. [Egg Prep - Bleaching]
Top up the Falcon tube to 14mL with M9.
?. [Egg Prep - Bleaching]
Repeat Steps 8 - 10 three more times, to thoroughly quench the bleach solution.
?. [Egg Prep - Bleaching]
After the final wash, top up to 10mL with M9.
?. [Egg Prep - Pooling L1s]
The next morning, transfer the newly hatched L1 larvae in 10mL M9 solution to a sterile conical Eppendorf tube using a glass Pasteur pipette.
?. [Egg Prep - Bleaching]
Pipette the 10mL solution onto an empty 60mm plate using a glass Pasteur pipette, and incubate at 20°C.
?. [Egg Prep - Pooling L1s]
Centrifuge for 2 minutes at maximum speed (14,000 rpm).
?. [Egg Prep - Pooling L1s]
Using a glass Pasteur pipette, remove as much supernatant as possible, and then pool all L1 worms together into a single Eppendorf tube.
?. [Egg Prep - Pooling L1s]
Dispense 3μL of L1 solution on the lid of a 60mm Petri plate, and count under microscope how many worms are in the droplet. Use this to estimate the total worm concentration of the Eppendorf.
?. [Egg Prep - Pooling L1s]
Using a DISTRIMAN Gilson repetitive pipette, dispense approximately 500 worms onto each plate.NB: The concentration may be adjusted by re-centrifuging and removing less supernatant with the Pasteur pipette (to dilute the solution if needed), and re-counting the number of worms in 3μL.
TITLE: Thermolabile Proteinase K Typical Reaction Protocol
AUTHORS: New England  Biolabs

[STEPS]
?. Reactions may be scaled-up linearly to accommodate larger amounts of substrate and larger reaction volumes. Optimal buffering reagents, enzyme quantity, incubation temperatures and times may vary for particular substrates. Typical restriction enzyme cleanup conditions are as follows:To a  restriction enzyme digest containing  of DNA and 10 units of a restriction enzyme:
50 µl
1 µg
?. Add  of Thermolabile Proteinase K, mix gently.
1 µl
?. Incubate reaction at  for .
37 °C
?. Incubate reaction at  for  to inactivate Thermolabile Proteinase K.
55 °C
Note: Optimal reaction buffers, reaction temperatures as well as additional reaction properties of Thermolabile Proteinase K can be found in detail in the FAQ. Optimization may be required for other cleanup applications (ligation, extension, PCR, etc.)
TITLE: Growth of mixed E. coli colonies 
AUTHORS: Wolfram Moebius

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Growth of mixed </span><span style = "font-style:italic;">E. coli</span><span> colonies on agar plates and between an agar plate and an agar pad</span></div></div>

[STEPS]
?. [Make plates]
Autoclave Lennox LB (10 g/l tryptone / casein digest peptone, 5 g/l NaCl, 5 g/l yeast extract) with 1.5 % agar (w/v).
?. [Make plates]
Optional: Add antibiotics at desired concentration after medium has cooled down sufficiently.
?. [Make plates]
Pipette  of LB and agar in Petri dishes with diameter of .
10 mL
?. [Make plates]
Leave plates plates to dry  at . Optionally enclose to avoid nonuniform drying.
4 Room temperature
?. [Store and prepare plates]
If not used the day after pouring plates, store at . Enclose in plastic container or bag to avoid further drying of plates.
4 °C
?. [Store and prepare plates]
Before usage of plates that have been refrigerated, warm up plates at .
37 °C
?. [Prepare bacterial cultures]
Grow overnight culture (colony picked or directly from glycerol frozen stocks) in Lennox LB.
?. [Inoculate colonies and incubate]
Mix  Lennox LB and  of each of the two overnight cultures in an Eppendorf tube.Vortex.
900 µl
50 µl
?. [Inoculate colonies and incubate]
Inoculate  in centre of plate and let dry.
1 µl
?. [Inoculate colonies and incubate]
Optional: Cover colonies with agar pad. Cut about  x  agar pad inside plate. Use spatula to lift pad. Place vertically next to colony and let fall upside down onto colony. Use spatula to remove bubbles by pressing on top.
?. [Inoculate colonies and incubate]
Place plates into container, together with sufficiently wet paper towels. Incubate for 7-8 days in a dark environment.
TITLE:  In vitro germination of Austropuccinia psidii urediniospores 
AUTHORS: Alyssa  M Martino, Rebecca M Degnan

[DESCRIPTION]
Optimisation of Austropuccinia psidii urediniospore germination for use in RNA extraction and cytogenetics. 

[STEPS]
SECTION: Spore collection, desiccation, and storage
1. Harvest fresh spores from heavily infected leaves by shaking into a paper bag.
SECTION: Spore collection, desiccation, and storage
2. Move fresh spores to a glass petri dish with no lid. Transfer petri dish to a desiccator with silica gel beads for 24 - 48 hours to dry the spores.


SECTION: Spore collection, desiccation, and storage
3. Sieve spores to remove any plant matter or dirt.
SECTION: Spore collection, desiccation, and storage
4. Aliquot spores into Nunc CryoTubes or similar for short or long term storage.

 


SECTION: Inocula preparation and plating
5. Create 2% water agar plates.
SECTION: Inocula preparation and plating
6. 
In a 15 mL centrifuge tube, make up inocula to a concentration of approximately 1 mg/mL in sterile distilled water (SDW) with 5ul/mL Tween 20 (0.05%). Mix thoroughly by gently inverting the tube several times.
 

SECTION: Inocula preparation and plating
7. Rehydrate spores in inocula for 30 minutes prior to use.
SECTION: Inocula preparation and plating
8. Pipette 2 uL aliquots of inocula onto agar plate, covering the entire plate with distinct 2 uL droplets and seal the plates with parafilm.
SECTION: Incubation
9. Germinate in incubator for a minimum of 8 hours in the dark at 18°C and 75% relative humidity.
SECTION: Determine Germination Percentage
10. For each aliquot, count the number of germinated spores under a dissecting microscope.  A germinated spore is considered to be a spore with a germination tube at least double the length of the urediniospore.
TITLE: Soft agar for yeast imaging 
AUTHORS: leonhard.bandilla  

[DESCRIPTION]
The here described medium can be used to image yeast without immobilization or compression while allowing for growth in all directions. The medium constists of YPD with agar added at a very low concentration to form a soft gel that allows for free growth in all directions. The media can be poured into any multiwell plate or dish to be imaged with an inverted microscope. This protocol is written for 1l of final medium but smaller quanities can be used.

[GUIDELINES]
When transfering the warm media to the final container, allow for enough height to image and for innoculation.
The final product is fragile and should not be tilted, shaken or inverted.

[STEPS]
1. To an autoclavable bottle add per 1l:
10 g Yeast extract
20 g Peptone
1 g Agar



3. Invert the bottle to dissolve everything apart from the agar

4. Autoclave or heat up sufficiently to dissolve the agar and insure sterility
5. Per 1l add 50 mL of sterile  40% glucose and mix, avoid introducing air bubbles

6. Let the solution cool down so that it can be safely handled and pour or pipette into the final container in which the imaging will take place. Avoid air bubbles as they can interfere with imaging. Make sure to avoid contamination by working near a bunsen burner or in a laminar flow hood
7. Wait for the agar solidify over night before innoculating the media. This media takes significantly longer to set, allow for multiple hours before any change in consistancy is noticible, be aware that the final product is very soft and can easily be mistaken for liquid media
2. Add 950 mL of water of appropriate quality per 1l of final media
TITLE: TissueCyte Installation And Alignment Guide
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol outlines some of the steps that are required to align a TissueCyte 1000 serial two-photon imaging system and install commonly replaced parts.</div><div class = "text-block"><span style = "font-weight:bold;">Note</span><span>: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.</span></div></div>

[STEPS]
TITLE: STRIP: Systematic Testing using Robotics and Innovation during Pandemics
AUTHORS: Peter H L Krijger, Tim A Hoek, Sanne Boersma, Lieke I P M Donders, Maaike M C Broeders, Mark Pieterse, Pim W Toonen, Ive Logister, Bram M P Verhagen, Marjon J A M Verstegen, Thomas W van Ravesteyn, Rene J T M Roymans, Francesca Mattiroli, Jo Vandesompele, Monique Nijhuis, Stefan Meijer, Anton van Weert, Edwin Dekker, Fred J Dom, Rob Ruijtenbeek, Lieven B J van der Velden, Jeroen H B van de Bovenkamp, Martijn Bosch, Wouter de Laat, Marvin E Tanenbaum

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">STRIP is a start-to-end streamlined and automated procedure for COVID-19 testing, centering on a single Tecan Fluent liquid-handling robot that can process over 14,000 samples per day. The sensitivity, specificity, and practical implementation of STRIP have been validated in a clinical study on 1128 individuals, meeting the standards set by the Dutch National Institute for Public Health and the Environment (Dutch CDC). Automation throughout the testing procedure dramatically reduces the workload of diagnostic laboratory personnel and potentially allows the placement of multiple STRIP liquid-handling robots per testing facility, further increasing testing capacity. The entire test procedure also requires only 3 pipet tips per sample, as well as reduced testing reagents due to process miniaturization, which is important given scarcity of testing consumables during the COVID-19 pandemic. Furthermore, STRIP is compatible with reagents from any supplier, and thus less sensitive to supply chain bottlenecks. Finally, the system is open and modular, facilitating adaptation of future developments in diagnostics. Overall, the system enabled substantial savings in personnel and reagents requirements compared with conventional diagnostic testing; when STRIP runs at full capacity, it is possible to rapidly recoup the initial outlay in the liquid-handling system from savings in personnel costs, reagents and materials.</div></div>

[STEPS]
TITLE: Preparation of Free Floating Coronal Mouse Brain Sections
AUTHORS: madalynn.erb

[DESCRIPTION]
This protocol details the preparation of free floating coronal mouse brain sections.

[STEPS]
SECTION: Collect Mouse Brain Tissue
1. Deeply anesthetize mice via intraperitoneal injection of 2X Avertin solution.
SECTION: Collect Mouse Brain Tissue
2. Perform transcardial perfusion using chilled saline solution
SECTION: Collect Mouse Brain Tissue
2.1. Keep 0.9% NaCl on ice.
SECTION: Collect Mouse Brain Tissue
2.2. Use approximately 60 mL saline solution per mouse.
SECTION: Collect Mouse Brain Tissue
3. Switch from saline solution to chilled PFA.
SECTION: Collect Mouse Brain Tissue
4. Remove brain immediately after PFA perfusion.
SECTION: Collect Mouse Brain Tissue
5. Incubate brain in PFA for 1440 min at 4 °C.
SECTION: Collect Mouse Brain Tissue
3.1. 4% paraformaldehyde in 0.1 Molarity (M) phosphate buffer (PB) pH 7.4.
SECTION: Collect Mouse Brain Tissue
3.2. Use approximately 60 mL PFA per mouse.
SECTION: Collect Mouse Brain Tissue
6. Transfer brains to 30% Sucrose / 0.1 Molarity (M) Phosphate Buffer (PB) – keep at 4 °C for ≥ 1440 min.
SECTION: Collect Mouse Brain Tissue
7. Tissue should be completely saturated with sucrose before sectioning.
SECTION: Collect Mouse Brain Tissue
7.1. Brains will sink to the bottom of the vial when saturated.
SECTION: Section Tissue
8. Use a Leica SM2010R Microtome.
SECTION: Section Tissue
8.1. Blade: Leica 16cm, knife angle set at 0 degree.
SECTION: Section Tissue
8.2. Cut thickness:  .
SECTION: Section Tissue
9. Adjust the microtome platform so that it is level with the blade and lock it into place.
SECTION: Section Tissue
10. Chill the microtome platform by covering it with crushed dry ice.
SECTION: Section Tissue
11. Apply 5-10 drops of 30% Sucrose / 0.1 Molarity (M) Phosphate Buffer (PB) solution to the chilled microtome platform and wait for it to solidify.
SECTION: Section Tissue
12. Use the microtome to gently shave the solidified sucrose to make a flat surface.
SECTION: Section Tissue
13. Use a razor blade to remove any spinal cord from the brain making a flat surface perpendicular to the rostral / caudal axis.
SECTION: Section Tissue
14. Apply 2-3 drops of sucrose to the existing sucrose platform and quickly position the brain with the olfactory bulbs pointing upwards.
SECTION: Section Tissue
14.1. Apply 2-3 more drops of sucrose to the top of the brain to securely freeze it to the microtome
platform.
SECTION: Section Tissue
15. Gently cover the brain in crushed dry ice to freeze the tissue.
SECTION: Section Tissue
16. Adjust the microtome platform so that the rostral / caudal axis is perpendicular to the blade and the dorsal / ventral axis is level with the blade.
SECTION: Section Tissue
17. Collect sections in a 24-well plate prefilled with cryoprotectant solution (0.1 Molarity (M) PB +30% Sucrose + 30% Ethylene Glycol).

Cryoprotectant solution
 

SECTION: Section Tissue
18. Seal plate with parafilm and store tissue at -20 °C.
TITLE: ImageJ Analyses of Mitochondria (HSP60) in TH+ cells
AUTHORS: Chiara Pavan, Stefano Frausin, Wai Kit (Leo) Lam, Clare Parish

[DESCRIPTION]
This protocol explains how to use Fiji to analyse mitochondria in iPSC derived dopaminergic neurons in culture for parameters such as network branching, sphericity and mean total branch length.

[BEFORE_START]
Requirements prior to analysis
-  Install Fiji
-  Locate images to analyse (HSP60+TH staining)
-  Install Mitochondria Analyzer Plugin (see references)

[STEPS]
SECTION: Image acquisition
1. Images analysed with this protocol were taken with Leica Stellaris 8 confocal (for mitochondrial morphology and ATG13-HSP60 co-localization, with FLIM 40x/1.30 oil immersion objective, HC PL
APO, CS2, using Leica Power HyD detector S and HyD detector X).

All mitochondria morphology images were acquired in 3D by optical sectioning under Lightning™ mode with a z-stack of 1.73μm and maximum voxel size of 94.7 nm laterally (x, y) and 350 nm axially (z). For each experimental sample 10 cells per sample were analyzed. 

Note: Deconvolved images were used for the mitochondrial morphology analysis.
SECTION: Analysis pipeline
2. 1. Open the image

 

2. In the TH channel (use the bar under the figure to go to the TH channel, here channel 2), use the Freehand Selection tool to mark the cell of interest
 
If the TH signal is not bright enough, go to Image>Adjust > color balance (only in the TH channel, don’t modify the other channels)

3. Save the region of interest selected by going to Analyze > Tool > ROI manager > Add [t]

4. Duplicate the HSP60 channel by clicking outside the selected ROI, then going to Image > Duplicate; select Channel 1 and tick hyperstack

5. Clear the staining outside the ROI by selecting the ROI (press on the ROI name on the ROI
manager panel), then going to Edit >Clear Outside >Yes

6. Convert the image to 8 bit by going to Image>Type>8bit

7. To optimize the Threshold, go to Plugins > Mitochondria Analyzer > 3D Threshold Optimize (for more info, see below) 
NOTE: This may take some time

 

8. Once decided the optimal values, go back to your picture (the one with only HSP60, duplicated, cleared), and apply the chosen parameters by going to Plugins > Mitochondria Analyzer > 3D Threshold and inserting the desired parameters.
NOTE: This may take some time
TITLE: a-commands and Allas
AUTHORS: Alise Ponsero

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Main interactions between Puhti and the Allas storage service at CSC using the a-command tools.</div><div class = "text-block"><span>For more detailed documentations on Allas and a-commands: </span><a href="https://docs.csc.fi/data/Allas/using_allas/a_commands" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://docs.csc.fi/data/Allas/using_allas/a_commands/</span></a></div></div>

[STEPS]
?. [Connecting to Allas]
In order to interact with Allas, especially to push and pull large files, it is best to request an interactive node.After login into Puthi, type the following to request an interactive node.sinteractiveFollow the instructions (choice of a project for the billing) and get an interactive node!
?. [Connecting to Allas]
Then we need to load and configure Allas.module load AllasThenAllas-confFollow the instructions (password and project name for billing units)
?. [list data stored in Allas]
To list all buckets stored in Allas, typea-listIf you want to display the content of a particular bucket, use the same function followed by the name of the bucketa-list my_bucket
?. [Push data to Allas]
In order to push data to Allas, we can use the command a-put.Navigate to the folder or file you want to push to Allas, and type:a-put my_file -b my_bucketIf the name of the bucket after the -b parameter is new, a new bucket with that name will be automatically created. You can push a complete folder using the same command:a-put my_folder -b my_bucketNOTE: If you are pushing a complete folder, it will be zipped before being sent to Allas. You won't be able to list the content of that folder anymore using the a-list function!
?. [get data back from Allas]
You can retrieve a file or folder stored in Allas using the a-get commanda-get my_bucket/my_fileIf you pushed a complete folder, you can retrieve the complete folder by the same methoda-get my_bucket/my_foldera-geta-geta-get
?. [get data back from Allas]
If you need to retrieve the whole bucket you have stored in Allas, you need a bit more work. You need first to list the content of the bucket using the a-list command and then read the list in a loop and download one by one the content.#make a list of objectsa-list my_bucket > list.txt#use the list in for loopfor ob in $(cat list.txt)do  a-get $obdone  #remove the object listrm list.txt