eamag/protocols-with-steps huggingface dataset:
TITLE: U Mass - Hepatic gluconeogenesis
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">Hepatic gluconeogensis is estimated using pyruvate tolerance test that measures systemic elevation of glucose partly derived from pyruvate and hepatic gluconeogensis following an intraperitoneal bolus injection of pyruvate in awake mice. Hepatic gluconeogenesis is affected by obesity and regulates glucose homeostasis.</div></div>

[STEPS]
?. Mice are fasted overnight (~15 hours) prior to the start of experiment.
?. Collect plasma sample (10 µl) before the start of experiment (basal-0 min) to measure basal glucose levels.
?. Administer intraperitoneal injection of pyruvate (1 g/kg body weight) using an insulin syringe.
?. Collect plasma samples (10 µl) at 10, 20, 30, 45, 60, 90, and 120 min following pyruvate injection to measure circulating glucose concentrations.
?. For data analysis, plasma glucose levels vs. time after pyruvate injection are plotted, and area-under-curve may be calculated to estimate hepatic gluconeogenesis.
TITLE: Mating assay
AUTHORS: Serena Ding

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">For imaging drug-treated young adult C. elegans in liquids using the Multiworm tracker. Worms are synchronised by picking L4s, and then the young adults are exposed to drugs for 4 hours prior to imaging for 15 mins in a liquid droplet on a coverslip mounted on a 3.5cm plate.</div></div>

[STEPS]
?. [Generating male stock (if needed) (~ -3 days) eg Friday]
Pick males from the heat-shocked plates onto the male stock plates, set up crosses with L4 hermaphrodites. If there are enough males, then set up 3 plates of 5 males x 2 hermaphrodites.
Keep repeating this step every 3-4 days to maintain male stocks throughout the experiment.
?. [Pre-pick L4 animals (-1 day PM)]
For each strain, pick ~150 L4 hermaphrodites onto 3 separate 55 mm plates (i.e. 50 animals per plate), and ~ 20 L4 males onto a single 55 mm plate.
?. [Imaging (-1 day PM)]
Seed each 35 mm imaging plates with 20 μL of undiluted OP50 in the centre of the plate. Let inoculate at room temperature over night.
Imaging plates should be about 1 week old and stored in the cold room prior to use
It's advisable to pre-aliquot the OP50 prior to the start of the experiment, to ensure batch consistency across different replicates.
?. [Imaging (Day 0 AM)]
Initialise the experimental set up on the computers with init_exp, open Gecko and adjust settings, etc.
?. [Imaging (Day 0 AM)]
Image for 15 minutes at 25fps with the .hdf5 file format.
?. [Imaging (Day 0 AM)]
Transfer the files to the server.
?. [Generating male stock (if needed) (~ -7 days) eg Monday AM]
Set the incubator to equilibrate at 30°C. Pick 6 L4 hermaphrodites onto a 55 mm plate, and do this 5 more times to have a total of 6 plates of 6 L4's. Incubate the L4's at 30°C for 6 hours and let recover at 20°C.
[heat shock at 30°C]
?. [Generating male stock (if needed) (~ -7 days) eg Monday AM]
Seed a few 55 mm male stock plates with a honey moon lawn of OP50 (small patch of bacteria to encourage mating).
It's not essential when this step is performed.
?. [Imaging (Day 0 AM)]
First identify and label plates in accordance with the experimental Excel template.
The plates will now be labelled with the set number and the rig position as well as half having a black dot annotation.
?. [Imaging (Day 0 AM)]
Use a wire or hair pick, transfer 25 adult hermaphrodites from Step 4 onto an imaging plate. Let animals acclimatise to their new environment for 25-30 min.
Place the animals away from the honeymoon lawn.
?. [Imaging (Day 0 AM)]
Use a wire or hair pick, transfer 1 adult male from Step 4 onto the imaging plate containing hermaphrodites from Step 7. Image immediately (ie no acclimation).
Place the animal away from the honeymoon lawn.
?. [Imaging (Day 0 AM)]
Hermaphrodite plates can be re-used if necessary, by removing the male (and the hermaphrodite it has mated with if she's possible to identify) and then adding a fresh male. There is no need to wait between male removal and addition, and image immediately after adding the new male.
TITLE: Trimble GPS Protocol
AUTHORS: Sabine St-Jean

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Here we describe the standardised protocol used by the </span><a href="http://www.caboscience.org/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Canadian Airborne Biodiversity Observatory</span></a><span> (CABO) to use the Trimble GPS with the NTRIP centimeter-level precision subscription from Cansel to record the precise location of a plot (corner or center) or a plant.</span></div></div>

[STEPS]
?. [GPS Connection]
Connect the Trimble antenna to your field cellphone, by plugging the cable coming out of the antenna into your cellphone charging port.
?. [GPS Connection]
Connect the external battery to the Trimble antenna, by plugging the USB port into the battery and the micro USB port into the antenna. Place the battery in its mount.
?. [GPS Connection]
Open the Trimble Mobile Manager app on your fieldwork cellphone.
?. [GPS Connection]
Sign in.
?. [GPS Connection]
Slide the Connect to Trimble Catalyst button.
?. [GPS Connection]
If the following screen shows up (it does not systematically), tick the Use by default for this USB accessory box.
?. [GPS Connection]
The app searching for GPS signal will show this screen.
?. [GPS Connection]
Wait for the precision to go down.
?. [Point Recording]
Open the Fulcrum app on your fieldwork cellphone.
?. [Point Recording]
In Fulcrum, go to the plot or tree record you want to georeference precisely, then go under Location.
?. [Point Recording]
Watch the value of Horizontal Accuracy and wait for it to stabilize.
DEFINITION OF A GOOD PRECISION:In forest environments, under a canopy with no leaves (i.e., after leaf fall), the precision can be under 30 cm.In the best conditions in an open environment, the precision can get down to 2 cm.
?. [Point Recording]
Once the Horizontal Accuracy is stable at a good precision, click Update Location with GPS. You’ll see the values next to Latitude, Longitude, Horizontal Accuracy and Altitude update.
?. [Point Recording]
Save your record.
TITLE: Gel Extraction
AUTHORS: NUS iGEM

[STEPS]
?. Add  Buffer QG into 2ml tube containing gel slice
450 µl
?. Incubate at  for
50 °C
?. Add  isopropanol to the sample and mix
150 µl
?. Transfer entire volume into QIAquick spin column
?. Centrifuge at  for
Centrifuge: 13000 33
?. Discard flow through
?. Add  Buffer PE to QIAquick spin column
750 µl
?. Centrifuge at  for
Centrifuge: 13000 33
?. Discard flow through
?. Repeat steps 7 - 9
?. Place spin column onto a new eppendorf tube
?. Elute DNA by adding  Buffer EB to the center of QIAquick membrane
30 µl
?. Incubate at  for
50 Room temperature
?. Centrifuge at  for
Centrifuge: 13000 33
?. Collect eluted DNA
TITLE: TIMSTOF Pro Crosslinking by Johannes Hevler; Albert Heck/ Richard Scheltema Labs
AUTHORS: Johannes Hevler 

[DESCRIPTION]
crosslinking.m:

Method to run cross-linking samples on TimsTof pro instrument. Method and energies are specifically
optimized for PhoX cross-linker reagent (by Richard Scheltema and Markus Lubeck (Brucker))

Standard_DDA_PASEF_1.1sec_cycletime_2segm_1st_15min_nospectra.m:

Method for classical bottom-up (proteomics) experiments. Optimized for LC systems that are operated 
without a trap and are euqipped with a 5 µL sample loop (flowrate 0.400 µL/min), as the Method is segmented: The
first 15 min of the method no spectra are saved. To further reduce the file size the noise filtering for Tims is turned on. (by Richard Scheltema and Markus Lubeck (Brucker))

[STEPS]
1. crosslinking.m:

Method to run cross-linking samples on TimsTof pro instrument. Method and energies are specifically
optimized for PhoX cross-linker reagent (by Richard Scheltema and Markus Lubeck (Brucker))

Standard_DDA_PASEF_1.1sec_cycletime_2segm_1st_15min_nospectra.m:

Method for classical bottom-up (proteomics) experiments. Optimized for LC systems that are operated 
without a trap and are euqipped with a 5 µL sample loop (flowrate 0.400 µL/min), as the Method is segmented: The
first 15 min of the method no spectra are saved. To further reduce the file size the noise filtering for Tims is turned on. (by Richard Scheltema and Markus Lubeck (Brucker))
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>RAW264.7 cells are a macrophage-like cell line derived from BALB/c mice.  These cells are loosely adherant and can be detached from cell culture flasks by light shaking or use of TrypLE (or Trypsin/EDTA).  </p>

[GUIDELINES]
<p>Only thaw cultures from frozen vials when there are no active cultures in the incubator.  Keep cells sterile by wearing gloves, ethanol treating your hands and surfaces, using sterile pipette tips, and <strong><em><u>only opening vials with cells in the BSL-2 laminar flow hood</u></em></strong>.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Model building, validation, and visualization
AUTHORS: Minghao Chen

[DESCRIPTION]
Model building, validation, and visualization

[STEPS]
SECTION: Model building
1. Generate a model by
[1] existing model in PDB
[2] creating a homology model by SWISS-MODEL
[3] creating a ab-initial model by AlphaFold
SECTION: Model building
2. Do the flexible model fitting by [ISOLDE]
Roughly fit the secodary structures into the map
SECTION: Validation
4. Validate the quality of the models by using the validation tools in Phenix and the online validation service provided by wwPDB (https://www.wwpdb.org/validation/validation-reports)

Make sure the statistics, particularly the Ramachandran plot, Rotamers, R.m.s. deviations and B-factors are resonable.
SECTION: Model building
3. Do model fitting manually in [Coot] or automatically in [Phenix]
Repeat the model fitting until the coordinates fit the map resonably 
SECTION: Visualization
5. Visualize the models and maps by [ChimeraX]
Create figures by [Adobe Illustrator]
Create movies by [iMovie]
TITLE: Gel electrophoresis
AUTHORS: Victoria Jackson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Gel electrophoresis</div></div>

[STEPS]
?. [Making the gel]
For  of a 1.2% agarose gel, add  of agarose to   TAE or TBE in a conical flask and mix by swirling the flask.
200 mL
2.4 g
200 mL
?. [Making the gel]
Microwave on a high power for about , or until boiling, swirling the flask approximately every  during heating to ensure all the agarose dissolves.
Use heat proof gloves.
?. [Making the gel]
Add  SafeView stain to the hot agarose and swirl to mix.
10 µl
?. [Making the gel]
Allow the agarose to cool until you are able to comfortably touch the outside of the flask (approximately ).
?. [Making the gel]
Pour the agarose into the casting tray. Pop any bubbles or move them to the edges of the mould using a pipette tip. Place the comb as desired.
?. [Loading the gel]
Place the gel (still in the casting tray) gently into the tank, ensuring there is enough TAE or TBE running buffer to cover it.
?. [Loading the gel]
On a piece of parafilm, dot  of 6x loading dye using a pipette.
1 µl
?. [Loading the gel]
Pipette  ladder or  PCR product directly onto the loading dye and mix by pipetting up and down several times.
5 µl
6 µl
?. [Loading the gel]
Load  into each well.
6 µl
?. [Running the gel]
Place the lid on the tank, making sure that the wells are at the negative electrode end of the tank. The samples should run from black to red (negative to positive).
?. [Loading the gel]
If the buffer used for PCR already contains loading dye, skip to step 11.
?. [Making the gel]
Allow the gel to set for approximately .
?. [Running the gel]
Set the power supply to between 80-120V, depending on the expected amplicon size and desired duration of electrophoresis.
?. [Running the gel]
Run the electrophoresis until the visible dye bands are about 75% of the way down the gel, approximately 1-1.5 hours.
Once complete, ensure you disconnect the power supply from the gel tank, before removing the lid.
TITLE: General procedure for long-term cryopreservation of Bacteria
AUTHORS: Andreas Sagen

[DESCRIPTION]
A simple and generalized procedure for freezing bacteria cells.

[STEPS]
SECTION: 50% Glycerol solution
1. Fill a 200 mL Duran bottle with 100 mL PBS

Materials:
 
SECTION: 50% Glycerol solution
2. Transfer 100 mL 100% Glycerol to Duran bottle

Materials:
 
SECTION: 50% Glycerol solution
3. Sterilize by autoclave at 121 °C for 15 min
4. Incubate a 20 mL batch of bacteria overnight

 

5. Aliquot saturated culture into an appropriate container. For example:
    60 µL saturated culture in a 200 µL reaction tube
    600 µL saturated culture in a 1.5 mL reaction tube
    900 µL saturated culture in a 2.0 mL cryotube
6. Mix in 50% glycerol solution. For example:
    40 µL saturated culture in a 200 µL reaction tube
    400 µL saturated culture in a 1.5 mL reaction tube
    600 µL saturated culture in a 2.0 mL cryotube
7. Transfer to an ultra-low temperature or vapor-phase nitrogen freezer for long-term storage
TITLE: PCR protocol for MgSTS marker genotyping
AUTHORS: Matthew Farnitano, Andrea Sweigart

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a standard PCR protocol used to amplify MgSTS markers from genomic DNA for genotyping. MgSTS markers were developed for use in Mimulus species and typically span length polymorphisms between conserved sequence regions. </div></div>

[STEPS]
?. [Prepare PCR reaction]
Determine the amount of reagents needed. The following amounts are sufficient for a single reaction:
[Molecular Biology Grade Water]
[5X GoTaq Flexi Buffer, colorless]
[MgCl2, 25mM stock]
[dNTPs, 2.5mM stock]
[BSA, 10 mg/mL stock]
[Premixed F+R Primers, 5uM stock]
[GoTaq G2 polymerase, 5 units/uL stock]
[DNA sample]
Note: Markers that are known to amplify in different, non-overlapping length ranges can be combined into a single PCR reaction. Add   of EACH premixed primer pair, and reduce your water volume accordingly so that the final reaction volume is   .
?. [Prepare PCR reaction]
Create a master mix with all ingredients except DNA. Add the polymerase last, mix well, and keep the mix on ice.
?. [Run PCR]
Run the following PCR cycler program:10 cycles of stepping down annealing temp:                >                > : Reduced by 1C every cycle to                 >35 cycles of constant annealing temp:                >                >                >
[2 mins 00 sec]
[0 mins 30 sec]
[0 mins 45 sec]
53 °C
[1 min 00 sec]
[0 mins 30 sec]
[0 mins 45 sec]
[1 min 00 sec]
[10 mins]
[Hold]
?. [Prepare genotyping reaction]
Once the PCR is complete, prepare the following reaction in a new plate:
HiDi and ROX size standard can be premixed and aliquoted beforehand. For marker lengths > 500, use ROX1000. For smaller lengths, ROX500 can be used.
[HiDi buffer]
[ROX size standard]
[PCR sample]
?. [Prepare genotyping reaction]
Briefly spin down the plate to remove air bubbles and sample stuck to the sides.Then cover with foil PCR covers and submit to your sequencing provider.
?. [Prepare PCR reaction]
Transfer  of master mix to each well of a PCR plate. Then add  sample DNA to each well. Add a plastic seal to the plate and press all edges to ensure the seal is tight. Mix well, spin the plate to collect droplets, and place in PCR machine.
8 µl
2 µl
?. [Prepare PCR reaction]
Thaw mix components except for GoTaq polymerase. Leave the polymerase in the freezer until it is needed.
TITLE: RNA concentration measurement and non-denaturing agarose gel electrophoresis
AUTHORS: Lisa-Maria Rosenthal, Giang Tong, Katharina Schmitt

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">RNA concentration measurement and non-denaturing agarose gel electrophoresis</div></div>

[STEPS]
?. •	Allow RNA to thaw on ice
?. •	Determine RNA concentration using a NanoDropTM 2000/2000c Spectrophotometers (Nucleic Acids – RNA mode, 2 µL sample size)
?. •	Determine RNA purity from protein contamination by assuring 260/280 ratio is 2.0
?. •	Determine RNA integrity and purity from genomic DNA contamination by electrophoresis. Load 400 ng RNA onto a 1% agarose gel with ethidium bromide (EtBr) in TBE buffer and run at 70 V until the dye line is approximately 75-80% of the way down the gel.
?. •	Visualize and document using a gel UV imaging/documentation system
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Phage lysis of host bacteria can be visualized by plaque formation on lawns of host cells in soft agar overlayed on agar plates (Adams, 1959; Sambrook et al. 1989). This principle can be used for the detection and subsequent isolation of specific lytic phages in environmental samples (e.g., Carlson 2005). Spotting environmental samples on lawns of a target host cell would thus reveal the presence of a lytic phage for that particular host cell in the given sample.

[GUIDELINES]
The main advantage of this procedure is that presence of lytic viruses is visually apparent as clearing zones on host bacterial lawns, and that subsequent isolation of phages is therefore fast, as the phages can be isolated directly from the plaques.

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: QMask Hemispheric Separation
AUTHORS: Michael X. Henderson

[DESCRIPTION]
This protocol describes QMask hemispheric separation.

[STEPS]
SECTION: QMask Hemispheric Separation
1. Open the Qmask tool and click “pick XML” and open the QuickN XML file generated in QuickNII.
SECTION: QMask Hemispheric Separation
2.  Enter the appropriate coordinates from the QMask Coordinates file.
SECTION: QMask Hemispheric Separation
3. Click “Destination” and navigate to the QVN\Mask folder. Click “Go”.
 
The mask output should be a black and White PNG that looks something like this:
 
SECTION: QMask Hemispheric Separation
4. Mask files shouldautomatically come with the appropriate naming convention with _mask at the end.
Example: NG18I_s061_mask
TITLE: Anesthetizing Sea Cucumbers
AUTHORS: Jon Eilers

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is for anesthetizing sea cucumbers. After soaking the sea cucumber in the menthol/ethanol/saltwaer solution for 30-60 minutes the sea cucumber should be flacid and relatively unresponsive to poking. No sea cucumber has eviscerated during surgery after being anesthetized although they do contract and appear like they are trying. The anesthestia wears off quickly after being placed back in salt water. </div><div class = "text-block">This protocol is based on the method for anesthetizing Apostichopus japonicus as published in the citation below</div></div>

[STEPS]
?. Add .5 grams of menthol crystals to 100 ml of ethanol and stir/shake until dissolved
?. Dilute to 10% menthol/ethanol solution by adding 900 ml of salt water
?. In a separate container, add 400 ml diluted menthol/ethanol solution and 600 ml saltwater to create a 40% diluted solution
?. Add sea cucumber to diluted solution and wait 30 minutes. After 30 minutes, gently poke and observe the reaction. If unresponsive, it is anesthetized. Otherwise, wait another 5-10 minutes and poke again.
Once anesthestized, the sea cucumber can be removed from the solution and will remain relatively unresponsive for 10-30 minutes.
If feeding tentacles are relaxed and hanging out of the mouth, it is anesthetized.
TITLE: Protein Extraction for Amyloid Beta Fractionation
AUTHORS: Lisa Blackmer-Raynolds, Tim Sampson

[DESCRIPTION]
This protocol can be used to isolate protein from murine brain tissue (volumes listed are for hippocampal samples) by solubility. It creates three protein fractions: tris soluble, triton soluble, and formic acid soluble. This protocol can be helpful when evaluating amyloid beta pathology as different amyloid beta structures can be found within each fraction. For example, insoluble amyloid beta associated with amyloid plaques will be found within the formic acid soluble fraction, whereas more soluble non-plaque associated amyloid beta will be found within the other two fractions.

[STEPS]
SECTION: Preparation
1. 5x Homogenization Buffer (store 4oC):
125 ml of 1.0 M Tris
30 ml of 0.5 M MgCl2
25 ml of 0.1 M EDTA (pH to 7.2 and store at 4°C for up to 6 months)
20ml of MiliQ Water
2. 1x Homogenization Buffer (dilute day of):
Dilute 5x Homogenization buffer to 1x in MiliQ Water and add 1 tablet protease inhibitor (cat#11697498001 Roche) per 10 mL

3. 1x Homogenization Buffer with 1% Triton X (dilute day of):
Add 1% Triton X-100 to 1x Homogenization buffer made above
4. 70% Formic Acid (store RT) **Caution Acidic**
7mL formic acid
3mL water
5. 1.5x Homogenization Buffer pH 14 (store 4oC) **Caution Corrosive**
Dilute 5x Homogenization buffer to 1.5x in MiliQ Water and pH to 14
SECTION: Tissue Processing
6. Weigh frozen tissue sample and record for later normalization
7. Homogenize the tissue on ice using quick bursts of a sonicator in 400µl 1x Homogenization buffer 
8. Centrifuge maximum speed at 4°C for 1hr
9. Collect supernatant as tris soluble fraction and store -80°C for later use
10. Homogenize the pellet on ice using quick bursts of a sonicator in 200µl 1x Homogenization Buffer with 1% Triton X-100
11. Centrifuge maximum speed at 4°C for 1hr
12. Collect supernatant as triton soluble fraction and store -80°C for later use
13. Homogenize the pellet on ice using quick bursts of a sonicator in 50µl 70% formic acid **Be careful with acid**
14. Centrifuge maximum speed at 4°C for 1hr
15. Collect supernatant as formic acid soluble fraction and neutralize with 1.5x Homogenization buffer pH14 on ice *Be careful with acid and base**. Store -80°C for later use
TITLE: Step-centrifugation Assay
AUTHORS: Caroline Brown, Snehasish Ghosh, Kallol Gupta

[DESCRIPTION]
This protocol details the protocol for conducting a step-centrifugation assay to determine the minimum required speed for full separation of vesicles from native nanodiscs.

[STEPS]
1. Resuspend cells in lysis buffer (50 millimolar (mM), 150 millimolar (mM) , 10 % volume ) and lyse using nitrogen cavitation (750 PSI for 15 min minutes).

2. Centrifuge lysed cells at 4000rpm for 10 minminutes to pellet cell debris.

3. Spin the supernatant in a series of sequential ultracentrifugation steps at speeds of   20,000xg, 100,000xg, 150,000xg, and 200,000xg with 60 min  hour for each spin.

4. After each spin subject the sample to dynamic light scattering to calculate population size distribution.
TITLE: Determination of IgA concentration by the Mancini test.
AUTHORS: Angel  Justiz-Vaillant

[STEPS]
?. An appropriate anti-IgA antiserum (antibody) is poured in the center well of an agar-containing plate.
?. Carefully circular wells are cut and detached from the plates.
?. A series of standards containing known concentrations of IgA are placed in separate wells, while “unknown” human serum samples and control are placed in other wells.
?. A ring of precipitate forms in the area of optimal antigen-antibody concentration, meaning anti-IgA - IgA reaction as the antigen diffuses radially.
?. The diameters of the rings are measured and perceived normally in 48-72 hours.
?. Finally, a standard curve is developed using the ring diameters of the standards versus the concentrations.
?. A curve is then used to plot the concentration of the control and unknown IgA samples.