eamag/protocols-with-steps huggingface dataset:
TITLE: UC Davis - Glucagon
AUTHORS: Peter Havel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Glucagon is a 29 amino acid polypeptide processed from proglucagon in pancreatic alpha cells. In intestinal L-cells proglucagon is cleaved into glicentin, corresponding to proglucagon residues no 1-69. Glicentin can further be processed into oxyntomodulin, corresponding to proglucagon residues no 33-69. These peptides are released simultaneously upon stimulation. Moreover, a fragment of glucagon corresponding to its Cterminal part (residues no 19-29), also designated mini-glucagon, is reported to be present in the pancreas in low amounts compared to the total glucagon content. </div><div class = "text-block">In general, glucagon has an effect opposite that of insulin, i.e. it raises blood glucose levels. It causes the liver to convert glycogen into glucose, which is then released into the blood stream. With longer stimulation, glucagon action in the liver results in a glucose-sparing activation of free fatty acid oxidation and production of ketones. During hypoglycaemia, glucagon secretion offers a protective feedback mechanism, defending the organism against damaging effects of glucose deficiency in the brain and nerves.</div></div>

[STEPS]
?. Prepare enzyme conjugate 1X solution and wash buffer 1X solution.
?. Prepare sufficient microplate wells to accommodate Calibrators, controls and samples in duplicate.
?. Pipette 10 µL each of Calibrators, controls and samples into appropriate wells.
?. Add 50 µL enzyme conjugate 1X solution to each well and attach the plate sealer.
?. Incubate on a plate shaker (700-900 rpm) over night (18-22h) at 2–8ºC
?. Wash 6 times with 700 µL wash buffer 1X solution per well using an automatic plate washer with overflow-wash function. After final wash, invert and tap the plate firmly against absorbent paper. Do not include soak step in washing procedure.Or manually, Discard the reaction volume by inverting the microplate over a sink. Add 350 µL wash solution to each well. Discard the wash buffer 1X solution, tap firmly several times against absorbent paper to remove excess liquid. Repeat 5 times. Avoid prolonged soaking during washing procedure.
?. Add 200 µL Substrate TMB.
?. Incubate on the bench for 30 minutes at room temperature (18–25ºC).
?. Add 50 µL Stop Solution to each well. Place plate on a shaker for approximately 5 seconds to ensure mixing.
?. Read optical density at 450 nm and calculate results. Read within 30 minutes.
TITLE: Microscopy-based GSH bead protein-protein interaction assay
AUTHORS: Minghao Chen, Xuefeng Ren

[DESCRIPTION]
Microscopy-based GSH bead protein-protein interaction assay

[STEPS]
1. Wash pre-blocked glutathione sepharose beads (GE Healthcare) with the reaction buffer (25 mM HEPES at pH 7.5, 150 mM NaCl, 1 mM MgCl2 and 1 mM TCEP) three times. 
2. Make a mixture of 1 μM purified GST tagged protein and 500 nM purified fluorescent protein in total 70 μl 



3. Incubate at Room temperature for 30 min, samples were mixed with additional 100 μL reaction buffer.




4. Transferred to the observation chamber for imaging. 



5. Acquire images on a Nikon A1 confocal microscope with a Nikon Plan APO VC 20x/0.75 NA UV Microscope Objective.
TITLE: Immunocytochemistry
AUTHORS: Michael Henderson, Naman Vatsa

[DESCRIPTION]
This is a protocol for immunocytochemistry for 24 and 96 well cell culture plates. 

[STEPS]
SECTION: Preparation
1. Prepare 1x phosphate buffer saline (PBS)
SECTION: Preparation
2. Prepare fixation solution: 4% paraformaldehyde/4% sucrose in PBS

 

SECTION: Preparation
3. Prewarm the fixative solution to 37 degrees Celsius 
SECTION: Preparation
4. Prepare blocking buffer: 3% Bovine Serum Albumin (BSA) in PBS
Seal the conical with parafilm to prevent evaporation and store at 4 degrees Celsius
SECTION: Preparation
5. Prepare 10% stock of Triton-X-100 in PBS
SECTION: Preparation
6. Prepare permeabilization buffer: 0.3% TX-100 in 3% BSA in PBS
SECTION: Staining
7. Thoroughly aspirate media from each well without disturbing cells
SECTION: Staining
8. Add pre-warmed fixative solution to the wells and incubate at room temperature for 15 minutes. (0.5 mL/well for 24-well plate, 100µL/well for 96-well plate).
SECTION: Staining
9. Rinse 5X with PBS. This and subsequent washes can be done with the plate washer for 96-well plates.
SECTION: Staining
10. Permeabilize neurons with a permeabilization buffer for 15 minutes at room temperature.
SECTION: Staining
11. Rinse 3X with PBS.
SECTION: Staining
12. Block neurons for a further 50 minutes with a blocking buffer at room temperature.
SECTION: Staining
13. Dilute primary antibodies in blocking buffer.
SECTION: Staining
14. Incubate cells with primary antibody for 2 hours at room temperature or overnight at 4°C.
SECTION: Staining
15. Rinse 5X with PBS.
SECTION: Staining
16. Dilute Alexa Fluor conjugated secondary antibodies corresponding to primary antibodies 
in blocking buffer in 1:500 dilution
SECTION: Staining
17. Incubate cells in secondary antibodies for 1 hour at room temperature.

 

SECTION: Staining
18. Rinse 5X with PBS.
SECTION: Staining
19. Mount coverslips onto glass slides with Prolong Gold mounting media. 96-well should be 
incubated with 100 µL DAPI 1:10,000 in PBS.
SECTION: Staining
20. Seal the 96-well plate with a black seal.
SECTION: Staining
21. Visualize staining on a fluorescent microscope.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
For use in "<a href="https://www.protocols.io/view/Obtaining-pure-cyanophage-stocks-liquid-assay-dqm5u5" target="_blank">Obtaining pure cyanophage stock: plaque purification</a>"

[GUIDELINES]
Use either the liquid amplification method (A) or the plate amplification method (B) in the "<a href="https://www.protocols.io/view/Obtaining-pure-cyanophage-stocks-liquid-assay-dqm5u5" target="_blank">Obtaining pure cyanophage stock: plaque purification</a>" protocol.

[STEPS]
?. 
?. 
?. 
?.
TITLE: Human Post Mortem Brain Processing
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the steps for processing post-mortem human brain, starting from whole brain or a single hemisphere.</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 U01MH114812. 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: Converting ssDNA oligos to dsDNA with T4 DNA polymerase
AUTHORS: Alex N Nguyen Ba

[DESCRIPTION]
This protocol allows one to convert ssDNA to dsDNA oligos. In principle, one can buy two complementary oligos and anneal them. However, there are a few cases where randomized bases are desired, and therefore complementary oligos can not be ordered. The protocol works similarly to PCR, except that the reaction is not performed at the high temperature of typical thermocycling, because the oligos are short and their melting temperature can be in the order of the extension temperature of high-fidelity polymerases.

[STEPS]
SECTION: Primer design
1. Design your target single-stranded DNA oligo such that the 3' end contains the reverse complement of an extending oligonucleotide. For example, appending "GTCATAGCTGTTTCCTG" to the end of your oligo will allow an oligonucleotide matching the M13 Reverse (-27) primer to extend it (5'-CAGGAAACAGCTATGAC-3').
SECTION: Oligo annealing
4. Mix in a PCR tube 5 µL of a 10 micromolar (µM) stock of the target oligo with 10 µL of a 10 micromolar (µM) stock of the extending oligo. Add 27ul of molecular biology grade water.

SECTION: Oligo annealing
5. Add 5 µL of  . Mix well by vortexing.

SECTION: Oligo annealing
6. Place the tube in a thermocycler and run the following protocol:
1. 95 degrees for 10 seconds
2. Decrease by 1 degrees
3. Repeat step 2 every 10 seconds, for 90 cycles.
4. Hold at 4 degrees.

SECTION: Primer design
2. Resuspend your oligonucleotides in 1/10th TE (10mM Tris, 0.1mM EDTA pH 8) to a final concentration of 100uM to form your stock oligonucleotide solutions.
SECTION: Primer design
3. Make working oligonucleotide solutions by diluting the stock oligonucleotides to a final concentration of 10uM.
SECTION: Oligo extension
7. Add 2.5 µL   (10 millimolar (mM) stock) and mix well by vortexing.
SECTION: Oligo extension
8. Go to the thermocycler and prepare the reaction cycle. When ready, start the protocol and pause when the block reaches 0 degrees.

1. 5 minutes at 0 degrees C.
2. 5 minutes at 22 degrees C.
3. 30 minutes at 37 degrees C.
4. Hold at 0 degrees.
SECTION: Oligo extension
9. Add 0.5 µL of   and mix well.

SECTION: Oligo extension
10. Place the tube in the thermocycler and unpause the run.
SECTION: Oligonucleotide cleanup
11. As soon as the reaction is completed, add   to 10 millimolar (mM).

SECTION: Oligonucleotide cleanup
12. Use a PCR purification kit to purify the oligo. Ensure the kit is able to purify short oligonucleotides if needed (a kit such as   can be suitable).
TITLE: protocol 10/06 1
AUTHORS: Maria Gul

[DESCRIPTION]
Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Integer in sapien. Praesent id justo in neque elementum ultrices. Cras elementum. Sed ac dolor sit amet purus malesuada congue. Nunc auctor. Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur? In convallis. Etiam quis quam. Aliquam ornare wisi eu metus. Curabitur bibendum justo non orci. Pellentesque sapien. Nullam at arcu a est sollicitudin euismod. Phasellus rhoncus. Maecenas libero. Praesent vitae arcu tempor neque lacinia pretium. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Phasellus faucibus molestie nisl. Vivamus ac leo pretium faucibus.

[STEPS]
1. Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Integer in sapien. Praesent id justo in neque elementum ultrices. Cras elementum. Sed ac dolor sit amet purus malesuada congue. Nunc auctor. Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur? In convallis. Etiam quis quam. Aliquam ornare wisi eu metus. Curabitur bibendum justo non orci. Pellentesque sapien. Nullam at arcu a est sollicitudin euismod. Phasellus rhoncus. Maecenas libero. Praesent vitae arcu tempor neque lacinia pretium. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Phasellus faucibus molestie nisl. Vivamus ac leo pretium faucibus.
2. Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Integer in sapien. Praesent id justo in neque elementum ultrices. Cras elementum. Sed ac dolor sit amet purus malesuada congue. Nunc auctor. Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur? In convallis. Etiam quis quam. Aliquam ornare wisi eu metus. Curabitur bibendum justo non orci. Pellentesque sapien. Nullam at arcu a est sollicitudin euismod. Phasellus rhoncus. Maecenas libero. Praesent vitae arcu tempor neque lacinia pretium. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Phasellus faucibus molestie nisl. Vivamus ac leo pretium faucibus.
3. Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Integer in sapien. Praesent id justo in neque elementum ultrices. Cras elementum. Sed ac dolor sit amet purus malesuada congue. Nunc auctor. Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur? In convallis. Etiam quis quam. Aliquam ornare wisi eu metus. Curabitur bibendum justo non orci. Pellentesque sapien. Nullam at arcu a est sollicitudin euismod. Phasellus rhoncus. Maecenas libero. Praesent vitae arcu tempor neque lacinia pretium. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Phasellus faucibus molestie nisl. Vivamus ac leo pretium faucibus.
TITLE: Tile Scan Imaging and Cell Counting
AUTHORS: Shiyi Wang

[DESCRIPTION]
Tile Scan Imaging and Cell Counting

[STEPS]
1. **Sample Preparation**
1.1. - Prepare 30 μm thick coronal sections containing the anterior cingulate cortex (ACC) and primary motor cortex (MOp) from P21 WT and LRRK2 G2019Ski/ki; Aldh1l1-eGFP mice.
2. **Confocal Microscopy**
2.1. - Acquire tile scan images using a confocal Leica SP8 STED microscope equipped with a galvo scanner and a 20x objective.
2.2. - Ensure consistent settings for image acquisition across all samples.
3. **Double Positive Cell Identification**
3.1. - Identify cells labeled with ALDH1L1-EGFP (astrocyte marker) and SOX9 (astrocyte marker) in the ACC and MOp regions.
3.2. - Use FIJI software with the cell counter tool for manual cell counting.
4. **Data Collection**
4.1. - Count ALDH1L1-EGFP and SOX9 double positive cells manually in 2-4 sections per brain.
4.2. - Analyze sections from 3 sex- and age-matched mice per genotype.
TITLE: Dip-C (Part 1: Chromosome Conformation Capture, for Fixed Nuclei)
AUTHORS: Longzhi Tan

[STEPS]
?. [Digestion]
Thaw a tube of fixed nuclei .
on ice
?. [Digestion]
Prepare 0.5% SDS ( per sample; recipe below for ): (final: ) water
50 µl
100 µl
5 µl
95 µl
?. [Digestion]
Resuspend nuclei in  0.5% SDS.
50 µl
?. [Digestion]
Incubate at  for .
62 °C
?. [Digestion]
Add: water
145 µl
25 µl
?. [Digestion]
Rotate at  for .
37 °C
?. [Digestion]
Add restriction enzyme and its buffer:
25 µl
20 µl
?. [Digestion]
Rotate at .
37 °C
?. [Ligation]
Centrifuge at .
Centrifuge: 1000 34, 5 min, 4 10
?. [Ligation]
Make Ligation Buffer ( per sample; recipe below for ): water
2 mL
1 mL
100 µl
5 µl
865 µl
?. [Ligation]
Remove supernatant leaving ~.
50 µl
?. [Ligation]
Resuspend in  Ligation Buffer.
1 mL
?. [Ligation]
Centrifuge at .
Centrifuge: 1000 34, 5 min, 4 10
?. [Ligation]
Remove supernatant leaving ~.
50 µl
?. [Ligation]
Resuspend in  Ligation Buffer.
1 mL
?. [Ligation]
Add .
10 µl
?. [Ligation]
Pipette to mix.
?. [Ligation]
Incubate at  for , occasionally inverting the tube.
16 °C
?. [Ligation]
Optionally filter with  or  to avoid clogging the flow cytometer.
?. [Ligation]
Aliquot if needed.
?. [Ligation]
Centrifuge at .
Centrifuge: 1000 34, 5 min, 4 10
?. [Ligation]
Remove supernatant.
?. [Ligation]
Store at .
-80 °C
?. [Flow Sorting]
On the day of flow sorting, thaw a tube of ligated nuclei  .
on ice
?. [Flow Sorting]
Resuspend in .
1 mL
?. [Flow Sorting]
Make 300 uM DAPI: 14.3 mM (5 mg/mL) DAPI (stock made by dissolving  in  water and stored at )
100 µl
2.1 µl
2 mL
4 °C
?. [Flow Sorting]
Add  300 uM DAPI (final: ).
1 µl
?. [Flow Sorting]
Flow sort single nuclei into either dry or containing lysis buffer (which requires lysis by incubation before storing; see Part 2 for details).
?. [Flow Sorting]
Proceed directly to Part 2, or store at .
-80 °C
?. [Flow Sorting]
Pipette to mix.
TITLE: How to extract cyanobacterial genome
AUTHORS: Leonardo Ken Okumura

[DESCRIPTION]
This protocol describes how to extract cyanobacterial genomes. The main treatments are carried out by sodium iodide, lysozyme, proteinase K and SDS.

[STEPS]
SECTION: Extracting cyanobacteria genome
1. Centrifuge the cyanobacteria culture at 16,000 x g for 10 minutes and remove the supernatant.
SECTION: Extracting cyanobacteria genome
2. Add 400 µL of Extraction Buffer and 100 µL of Saturated NaI Solution to the pellet, and mix well.
SECTION: Extracting cyanobacteria genome
3. Incubate at 37°C for 30 minutes.
SECTION: Extracting cyanobacteria genome
4. Centrifuge at 177,000 x g for 5 minutes and remove the supernatant.
SECTION: Extracting cyanobacteria genome
5. Add 100 µL of Extraction Buffer to the pellet and mix. Then centrifuge at 177,000 x g for 5 minutes and remove the supernatant.
SECTION: Extracting cyanobacteria genome
6. Add 500 µL of Extraction Buffer with Lysozyme to the pellet and mix.
SECTION: Extracting cyanobacteria genome
7. Incubate at 37°C for 2 hours.
SECTION: Extracting cyanobacteria genome
8. Add 5 µL of Proteinase K Solution and 15 µL of SDS, and mix.
SECTION: Extracting cyanobacteria genome
9. Incubate at 37°C overnight.
SECTION: Extracting cyanobacteria genome
10. Add 125 µL of SDS, and mix.
SECTION: Extracting cyanobacteria genome
11. Incubate at 37°C for more than 3 hours.
SECTION: Extracting cyanobacteria genome
12. Add an equal volume of PCI and gently rotate for 5 minutes.
SECTION: Extracting cyanobacteria genome
13. Centrifuge at 4,000 rpm for 10 minutes at room temperature, and transfer the supernatant to a new tube.
SECTION: Extracting cyanobacteria genome
14. Add 3 µL of RNase A, and mix.
SECTION: Extracting cyanobacteria genome
15. Incubate at 37°C for 30 minutes.
SECTION: Extracting cyanobacteria genome
16. Repeat steps 12 and 13.
SECTION: Extracting cyanobacteria genome
17. Add isopropanol in more than equal volume and mix.
SECTION: Extracting cyanobacteria genome
18. Centrifuge at 16,000 x g for 1 minute at 4°C and remove the supernatant.
SECTION: Extracting cyanobacteria genome
19. Repeat steps 17 and 18, replacing isopropanol with 70% ethanol.
SECTION: Extracting cyanobacteria genome
20. Air-dry the pellet and resuspend in distilled water.
TITLE: Create a class Github repository
AUTHORS: Bonnie Hurwitz, Ken Youens-Clark

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The github repository will serve as a location for you to turn-in your homework, and allow the instructors to push back code modifications and suggestions.</div></div>

[STEPS]
?. Login to http://github.com
?. Fork (or "copy") the class repository into your own Github account. This creates a copy of the repository in your own account that you can edit and update. Select your Github account as the place to "fork" the repository.
?. Click on your "biosys-analytics" repository. Click on the "Settings" tab on the top bar, click on the "Collaborators" tab on the side bar, and search for & add the instructors as collaborators. The instructors Github usernames are: bhurwitz33 and kyclark.
?. Go to the class Github repository: https://github.com/hurwitzlab/biosys-analytics
?. Go to your Github account. You will see a directory called biosys-analytics. This is your copy.http://github.com/my-username . (replace my-username with yours!)
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>L1 trace element solution, modified from Bigelow: NCMAM Center. Components to prepare 1 L of trace element solution.</p>

[BEFORE_START]
<p>Prepare primary stock solutions (in dH<sub>2</sub>O) of the following:</p>
<ul>
<li>MnCl<sub>2</sub>·4H<sub>2</sub>O: 178.10 g/L</li>
<li>ZnSO<sub>4</sub>·7H<sub>2</sub>O: 23.00 g/L</li>
<li>CoCl<sub>2</sub>·6H<sub>2</sub>O: 11.90 g/L</li>
<li>CuSO<sub>4</sub>·5H<sub>2</sub>O: 2.50 g/L</li>
<li>Na<sub>2</sub>MoO<sub>4</sub>·2H<sub>2</sub>O: 19.9 g/L</li>
<li>H<sub>2</sub>SeO<sub>3</sub>: 1.29 g/L</li>
<li>NiSO<sub>4</sub>·6H<sub>2</sub>O: 2.63 g/L</li>
<li>Na<sub>3</sub>VO<sub>4</sub>: 1.84 g/L</li>
<li>K<sub>2</sub>CrO: 1.94 g/L</li>
</ul>
<p>Filter sterilize individual solutions through a 0.2 micron PES membrane.</p>
<p>Store frozen in 1 mL aliquots.</p>

[GUIDELINES]
<p>First, prepare primary stock solutions. Glassware should be acid-washed and rinsed well with dH<sub>2</sub>O before use. If unable to measure small quantities accurately, either scale up the recipe or prepare stock solutions of the individual compounds.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: WC Medium
AUTHORS: Richard W Lambrecht

[DESCRIPTION]
Protocoll description to prepare WC medium.

[STEPS]
SECTION: Stock Solution preparation
1. The stock solutions don't need to be autoclaved after preparation. Make sure to use volumetric flasks to ensure the right concentrations of the salts and transfer the solution to autoclavable flasks and store it in the fridge. Remember to start the preparation with less than 1L of MilliQ water, and after adding the salts complete the volume to 1L.
SECTION: Stock Solution preparation
1.1. Stock solution A: 36,80g of (CaCl2+2H2O) in 1L of MilliQ water
SECTION: Stock Solution preparation
1.2. Stock solution B: 37g of (MgSO4+7H2O) in 1L of MilliQ water
SECTION: Stock Solution preparation
1.3. Stock solution C: 12,60g of (NaHCO3) in 1L of MilliQ water
SECTION: Stock Solution preparation
1.4. Stock solution D: 11,40g of (K2HPO4 +H2O) in 1L of MilliQ water
SECTION: Stock Solution preparation
1.5. Stock solution E: 85 g of (NaNO3) in 1L of MilliQ water
SECTION: Stock Solution preparation
1.6. Stock solution F: 21,20 g of (Na2SiO3 + 5H2O) in 1L of MilliQ water
SECTION: Stock Solution preparation
1.7. Stock solution G (Supplement elements): add the following salts in 1L of MilliQ water:
- 4,36 g of (Na2EDTA)
- 3,15 g of (FeCl3 + 6H2O)
- 0,010 g of (CuSO4 + 5H2O)
- 0,022 g of (ZnSO4 + 7H2O)
- 0,010 g of (CoCl2 + 6H2O)
- 0,180 g of (MnCl2 + 4H2O)
- 0,006 g of (Na2MoO4 + 2H2O)
- 1,0 g of (H3BO3)
SECTION: Stock Solution preparation
1.8. Stock solution H (Vitamin Mix): add the following in 1L of MilliQ water:
- Thiamine hydrochloride (Vitamin B1): 0,1 g of (C12H17ClN4OS)
- Biotin: 0,0005 g of (C10H16N2O3S)
- Cyanocobalamin (Vitamin B12): 0,0005 g of (C63H88CoN14O14P)


Aliquot the solution in 15 mL falcon tubes, and freeze them until used at -20 ºC.
SECTION: Medium preparation (for 1L of WC)
2. Weigh 0,115 g of TES Buffer (C6H15NO6S) and add it to a volumetric flask with almost 1L of MilliQ water
SECTION: Medium preparation (for 1L of WC)
3. Add 1 mL of each of the stock solutions A to G to the medium preparation bottle.
SECTION: Medium preparation (for 1L of WC)
4. Complete the volume until 1 liter with MilliQ water
SECTION: Medium preparation (for 1L of WC)
5. Transfer the prepared medium to an autoclavable flask, labeled with autoclaving tape.
 On the label put the information about the liquid contained (WC medium), the date, the person responsible for the preparation, and a box to check when adding the solution H (Vitamin Mix). 
SECTION: Medium preparation (for 1L of WC)
6. Autoclave the medium at 121ºC and let it cool down to room temperature.
SECTION: Medium preparation (for 1L of WC)
7. In a clean bench: syringe filter 1 mL of the defrosted solution H (Vitamin Mix), add it to the medium (1 ml per Liter), and check the box on the tape.
TITLE: Motor behavioral evaluation of mice
AUTHORS: Tae-Un Han

[DESCRIPTION]
This is a general protocol to test motor coordination of mice used models of neurological disorder

[STEPS]
SECTION: Rotarod test
1. On first day, mice are acclimated to standing on a non-rotating rod (Rotamex 5, Columbus Instrument) for three 1 min trials with 10 min interval. 

SECTION: Rotarod test
2. Three hours later, mice are acclimated to walking on the rod over three 90 s  trials at a speed of 4 rpm

SECTION: Rotarod test
3. On next day, latency of mice staying in the rotarod is recorded with accelerating speed of 4-40 rpm increasing by 1 rpm every 8.3 seconds for a maximum of 5 min  per trial. 

SECTION: Rotarod test
4. Repeat experiment for three trials daily for two days
SECTION: Beam walk test
8. For actual experiment, mice crosses both beams twice per trial  for three trials win 5 minresting periods in between trials

SECTION: Beam walk test
5. Mice are trained to cross beams of 1~1.5m length of round beam.  24mm and 18mm diameter of beams are used.
SECTION: Beam walk test
6. Beam-crossing training is done for three times for two days
SECTION: Beam walk test
7. During the experimental day,  its motor coordination will be measured by determining the number of slips, the latency required to cross the beam as the mouse traverses the beam.