eamag/protocols-with-steps huggingface dataset:
TITLE: MTT (Assay protocol
AUTHORS: Abdulkareem Hameed Abd, Enas Jawad Kadhim, Matin Mahmood

[DESCRIPTION]
MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide)) is used to assess cell viability as a function of redox potential. Actively respiring cells convert the water-soluble MTT to an insoluble purple formazan.

AMJ13: 2 (Ahmed.Murtuda ,Jabria 2013)cell line:
The cell line of breast cancer has been obtained from Iraqi breast cancer which originated from the prime tumor of an old Iraqi woman (70 years) with a histological identification with carcinoma of infiltrating ductal (1).

SK-GT-4:esophageal carcinomacell line was established from a primary tumors in 1989 from a 89 year-old Caucasian male who presented with dysphagia secondary to a well-differentiated adenocarcinoma arising in the Barrett epithelium of the distal oesophagus 

[STEPS]
SECTION: Maintenance of Cell lines
1. SK-GT-4 cell line, was maintained in MEM supplemented with 10% Fetal bovine, 100 units/mL penicillin, and 100 µg/mL streptomycin. Cells were passaged using Trypsin-EDTA reseeded at 50% confluence twice a week, and incubated at 37 °C 
SECTION: MTT Assay
4. Cell lines were seeded at 1 × 104cells/well. After 24 hrs. or a confluent monolayer was achieved, cells were treated with tested compound
SECTION: MTT Assay
5. Cell viability was measured after 72h of treatment by removing the medium
SECTION: MTT Assay
6. adding 28 µL of 2 mg/mL solution of MTT
SECTION: MTT Assay
7. incubating the cells for 1.5 h at 37 °C.
SECTION: MTT Assay
8. After removing the MTT solution, the crystals remaining in the wells were solubilized by the addition of 130 µL of DMSO (Dimethyl Sulphoxide) 
SECTION: MTT Assay
9. followed by 37 °C incubation for 15 min with shaking (orbital shaker)
SECTION: MTT Assay
10. The absorbency was determined on a microplate reader at 492 nm (test wavelength)
SECTION: MTT Assay
11. The assay was performed in triplicate
SECTION: MTT Assay
12.  The inhibition rate of cell growth (the percentage of cytotoxicity) was calculated as the following equation
  

SECTION: MTT Assay
3. To determine the cytotoxic effect, the MTT cell viability assay was conducted on 96-well plates
SECTION: Maintenance of Cell lines
2. NHF cell line, was maintained in MEM supplemented with 10% Fetal bovine, 100 units/mL penicillin, and 100 µg/mL streptomycin. Cells were passaged using Trypsin-EDTA reseeded at 50% confluence twice a week, and incubated at 37 °C
TITLE: HPLC Analysis of Nucleotides
AUTHORS: Xuefeng Ren, Annan SI Cook

[DESCRIPTION]
Ion-pair reversed phase HPLC analysis of the nucleotide bound to PI3KC3-C1.  

[STEPS]
SECTION: Denaturation of PI3KC3-C1
1. Heat the PI3KC3-C1 sample in a heat block at 90 °C for 10 min to denature the protein.
SECTION: Centrifugation
2. After denaturation, centrifuge the sample at 21000 rpm, 15 min to pellet any precipitated PI3KC3-C1.
SECTION: Supernatant Transfer
3. Carefully transfer the supernatant containing the released nucleotide to a clean tube, leaving behind the pellet.
SECTION: Preparation of Nucleotide Standards
4. Prepare 10 micromolar (µM) stock solutions of ATP, ADP, GTP, and GDP standards in 25 millimolar (mM) HEPES pH 7.5, 150 millimolar (mM) NaCl, 2 millimolar (mM) MgCl2, and 2 millimolar (mM) TCEP.
SECTION: HPLC Column Equilibration
5. Equilibrate the C18 reverse-phase HPLC column with the mobile phase Buffer A ( and Buffer B according to the manufacturer's instructions.
SECTION: HPLC Gradient Program
6. Set up the HPLC system with the following parameters:

Mobile phase: gradient of Buffer A to Buffer B.
Gradient duration: 30 min per run.
Wavelength for detection: 254 nm.
SECTION: Sample Injection
7. Inject 60 µL each of the nucleotide standards and the Protein nucleotide into the HPLC system.
SECTION: Data Collection
8. Allow the HPLC system to run the samples through the column.
SECTION: Data Collection
9. Record the retention times for each eluted nucleotide as they appear in the chromatogram.
SECTION: Analysis
10. Compare the retention times of the eluted nucleotides in the sample to those of the known standards (ATP, ADP, GTP, GDP).
SECTION: Analysis
11. Identify the bound nucleotide(s) based on their retention times in the chromatogram.
TITLE: Protein extraction protocol
AUTHORS: rabdelha  

[DESCRIPTION]
This protocol details protein extraction procedure.

[STEPS]
SECTION: Protein extraction protocol
1. Warm reagent to Room temperature.

SECTION: Protein extraction protocol
2. Add protease inhibitor tablet (1 tablet/10 mL) (complete EDTA free).

SECTION: Protein extraction protocol
3. Add amount of reagent to each sample and transfer to bead beating tube.
SECTION: Protein extraction protocol
4. Homogenize tissue sample in bead beater (see protocol in notebook), speed 3; let samples sit on ice for 10-15 min after to settle.
SECTION: Protein extraction protocol
5. Centrifuge homogenized sample at 10000 rpm for 5 min.

SECTION: Protein extraction protocol
6. Collect supernatants and store in -80 °C.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol provides a method for predicting the locations of the open reading frames (ORFs) using the Glimmer3 toolkit. Methods based on the publication: <br /><br />Hannigan, Geoffrey D., et al. "The Human Skin Double-Stranded DNA Virome: Topographical and Temporal Diversity, Genetic Enrichment, and Dynamic Associations with the Host Microbiome." <em>mBio</em> 6.5 (2015): e01578-15.

[BEFORE_START]
Supplementary information available at:<br /><br /><a href="https://figshare.com/articles/The_Human_Skin_dsDNA_Virome_Topographical_and_Temporal_Diversity_Genetic_Enrichment_and_Dynamic_Associations_with_the_Host_Microbiome/1281248" target="_blank">https://figshare.com/articles/The_Human_Skin_dsDNA_Virome_Topographical_and_Temporal_Diversity_Genetic_Enrichment_and_Dynamic_Associations_with_the_Host_Microbiome/1281248</a>

[GUIDELINES]
<strong>Required Software:</strong><br />
<ul>
<li>Glimmer3 v3.02</li>
</ul>
<strong>Relevant Files</strong><br />
<ul>
<li>Output: Virome_ORFs/Contigs_no_block_with_names_glimmer_output_final.fa</li>
</ul>
<br />After the <a href="https://www.protocols.io/view/Script-P2-Contig-Assembly-ed9ba96" target="_blank">contigs were generated</a> from the concatenated fasta files, we predicted the locations of the open reading frames (ORFs) and extracted them from the contigs using the Glimmer3 toolkit. First we generated the needed directories and copied the contig files over to a new directory.<br /><br />We then built an Interpolated Context Model (ICM), used it to predict the open reading frames, and extracted the open reading frames from the contig sequences. Finally we removed the block fasta format of the ORFs because this can interfere with some of our downstream analyses.

[STEPS]
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TITLE: Restriction Digest
AUTHORS: Zhujun Wei

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol includes three different methods of digestion.</div></div>

[STEPS]
?. Add the following reagents to a tube.   ABCD1Total10ul30ul50ul2DNA0.4ug1.2ug2ug3Enzyme0.2ul0.6ul1ul4Buffer(10*)1ul3ul5ul5ddwater      Fill the rest with water
ABCD1Total10ul30ul50ul2DNA0.4ug1.2ug2ug3Enzyme0.2ul0.6ul1ul4Buffer(10*)1ul3ul5ul5ddwater      Fill the rest with water
?. Pipette up and down thoroughly.
?. Add the following reagents to a tube.   ABCD1Total10ul30ul50ul2DNA0.4ug1.2ug2ug3Enzyme0.2ul0.6ul1ul4Buffer(10*)1ul3ul5ul5ddWaterFill the rest with waterFill the rest with waterFill the rest with water
ABCD1Total10ul30ul50ul2DNA0.4ug1.2ug2ug3Enzyme0.2ul0.6ul1ul4Buffer(10*)1ul3ul5ul5ddWaterFill the rest with waterFill the rest with waterFill the rest with water
?. Put at 37℃ for about 2 hours (according to different enzymes).
?. Pipette up and down thoroughly.
?. Put at 37℃ for about 2 hours (according to different enzymes).
?. Put at 65℃ for about 20 min.(according to different enzymes)
?. Purification of enzyme digestion products with Cycle Pure Kit D6492（OMEGA bio-tek）
?. DNA product concentrations were measured using Nano
?. Add the following reagents to a tube:   ABCD1Total10ul30ul50ul2DNA product0.4ug1.2ug2ug3CIP(NEB)0.2ul0.6ul1ul4Cutsmart(10*)1ul3ul5ul5ddWaterFill the rest with waterFill the rest with waterFill the rest with water
ABCD1Total10ul30ul50ul2DNA product0.4ug1.2ug2ug3CIP(NEB)0.2ul0.6ul1ul4Cutsmart(10*)1ul3ul5ul5ddWaterFill the rest with waterFill the rest with waterFill the rest with water
?. Pipette up and down thoroughly.
?. Put at 37℃ for about 2 hours (according to different enzymes).
?. Add the following reagents to a tube.   ABCD1Total10ul30ul50ul2DNA0.4ug1.2ug2ug3Enzyme 10.2ul0.6ul1ul4Enzyme 20.2ul0.6ul1ul5Buffer(10*)1ul3ul5ul6ddwaterFill the rest with waterFill the rest with waterFill the rest with water
ABCD1Total10ul30ul50ul2DNA0.4ug1.2ug2ug3Enzyme 10.2ul0.6ul1ul4Enzyme 20.2ul0.6ul1ul5Buffer(10*)1ul3ul5ul6ddwaterFill the rest with waterFill the rest with waterFill the rest with water
?. Pipette up and down thoroughly.
?. Put at 37℃ for about 2 hours (according to different enzymes).
?. Add the following reagents to a tube.   ABCD1Total10ul30ul50ul2DNA product0.4ug1.2ug2ug3DpnI(NEB)0.2ul0.6ul1ul4Cutsmart(10*)1ul3ul5ul5ddwaterFill the rest with waterFill the rest with waterFill the rest with water
ABCD1Total10ul30ul50ul2DNA product0.4ug1.2ug2ug3DpnI(NEB)0.2ul0.6ul1ul4Cutsmart(10*)1ul3ul5ul5ddwaterFill the rest with waterFill the rest with waterFill the rest with water
?. Pipette up and down thoroughly.
?. Put at 37℃ for about 2 hours (according to different enzymes).
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The main purpose of pregnancy testing as part of a clinical research protocol is to minimize risk to an embryo or fetus from exposure to potentially harmful study interventions.  The specific goal of testing may be to prevent exposure completely (by testing prior to any study interventions), or to minimize the duration of exposure during early pregnancy (by periodic testing during the study).</p>
<p> </p>
<p> </p>
<p>This survey provided five clinical study scenarios and asked respondents a series of questions about the acceptable risk of an unintended fetal exposure to a drug, the importance of several factors going into clinical study design, and the type of pregnancy testing protocol that you would recommend.</p>

[STEPS]
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is modified from Wiegand and colleagues protocol<sup>1</sup>  to fit the conditions of our experiments. </p>
<p> </p>
<p><sup>1</sup>Wiegand, Irith, Kai Hilpert and Robert E W Hancock (2008). \Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances". I: Nat Protoc 3.2, s. 163{75</p>

[STEPS]
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?. 
?. 
?. 
?. 
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?. 
?. 
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?. 
?. 
?.
TITLE: Transformation electroporation
AUTHORS: Elizabeth Fozo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Preparation of component cells and their transformation by electroporation</div></div>

[STEPS]
?. [Preparation of component cells]
Inoculate a single colony into appropriate media with antibiotics as needed. Grow overnight at the appropriate temperature and shaking conditions.
?. [Preparation of component cells]
Dilute ON culture back to an optical density of 0.01 in the morning in 25 mL of cells for every two transformations (4 transformations would be 50 mL).
?. [Preparation of component cells]
row at appropriate conditions until OD600 is ~0.4 to 0.6. Do not grow past this point.
?. [Preparation of component cells]
Pour cells into 50 mL conicals. Spin down cells at 3500 RPM at 4°C for 10 minutes (program 1 in swinging bucket centrifuge)
?. [Preparation of component cells]
Resuspend pellet in an equal volume of ice-cold water as the amount of culture placed in the conical. Spin cells down again.
?. [Preparation of component cells]
For every 25 mL of culture, resuspend pellet in 800 µL of ice-cold water, place into Eppendorf tube that has been chilled on ice.
?. [Preparation of component cells]
Spin down cells at 13,000 RPM at 4°C for 3 minutes.
?. [Preparation of component cells]
Aspirate supernatant.
?. [Preparation of component cells]
Resuspend pellet in 100 µL ice-cold water per 25 mL of culture.
?. [Transformation by electroporation]
Place the appropriate number of gene pulse cuvettes and Eppendorf tubes on ice in advance.
?. [Transformation by electroporation]
To the chilled Eppendorf tube, mix 40-50 µL of competent cells, and the appropriate amount of ligation or plasmid DNA.
?. [Transformation by electroporation]
Transfer to a chilled cuvette; make sure you tap so there are no air bubbles!
?. [Transformation by electroporation]
Wipe off any water/ice from the cuvette.
?. [Transformation by electroporation]
Electroporate using EC 2 on electroporator.
?. [Transformation by electroporation]
Immediately place back on ice; read the time constant and note this in your notebook.
?. [Transformation by electroporation]
Once complete, add 0.5 mL SOC media to rescue cells.
?. [Transformation by electroporation]
Transfer cell mixture to a 14 mL snap-cap tube.
?. [Transformation by electroporation]
Recover at appropriate growth conditions for 1 hour.
?. [Transformation by electroporation]
For ligations, transfer recovery to an Eppendorf tube and spin down at 13000 RPM for 5 minutes. Resuspend pellets in 100 µL and plate on the appropriate plate with appropriate antibiotics.
?. [Transformation by electroporation]
For whole plasmids, plate a dilution series. 10-3 to 10-6 suggested.
?. [Transformation by electroporation]
Incubate overnight at the appropriate temperature.
TITLE: Rapid and direct method to extract SARS-CoV-2 RNA from municipal wastewater using the Chemagic™ 360 12-rod head platform
AUTHORS: Adrian A Vasquez, Nicholas W West, Azadeh  Bahmani, Jeffrey   L. Ram

[DESCRIPTION]
Wastewater based epidemiology (WBE) has emerged as a strategy to identify, locate, predict, and manage outbreaks of COVID-19, as an early warning signal to public health authorities of an expected surge in cases that may overwhelm local and global health care resources.. The WBE process is based on assaying municipal wastewater for molecular markers of the SARS-CoV-2 virus. The standard process for sampling municipal wastewater is time-consuming and requires the handling of large quantities of wastewater, which negatively affects throughput and timely reporting, and can increase safety risks. We report on a rapid and direct mostly automated method to assay multiple sub-samples of a bulk wastewater sample using a 75 minute run on the Chemagic™ 360 12 rod head platform. Including a preceding setup and incubation step, twelve 10 ml samples can be processed to purified RNA in 2.5 hrs. Up to 10 ml of wastewater from 12 different collection sites can be processed in 2.5 hrs.

[STEPS]
1. Transfer 45 ml45 mL  from wastewater sample to 50 ml conical tube.

2. Centrifuge the 45 ml wastewater for 15 minutes at 5000 RPM (Thermo Scientific Sorvall ST 16R centrifuge, Waltham, MA).
     

3. Remove 10 ml10 mL  from the spun down 50 ml conical tube and transfer to 50 ml conical tube containing master mix of the following solution:
7 µl Poly A RNA (CMG842)
50 µl Proteinase K (CMG749)

4. Add 8 ml8 mL  lysis buffer 1 (CMG749) to the 50 ml conical tube containing the wastewater + master mix.

5.  Incubate on a dry bath (USA Scientific, Ocala, Fl) for 30 minutes at 55 °C.
   

6. Add 50 µl50 µL  resuspended magnetic beads (CMG749) to the 50 ml conical tube and put the 50 ml conical tube in Chemagic 360™ that is prepared with empty 50 ml conical tubes and Sarstedt® tubes with 100 µl100 µL  elution buffer.
  
 

7. Run Chemagic 360™ viral protocol (chemagic™Viral10k 360 H12 prefilling drying VD210119.che) for 75 mins.
  
 

8. Remove Sarstedt® tubes and transfer eluted RNA to new microcentrifuge tubes for long term storage at -80 C°.
TITLE: Transformation of Chemically Competent (Smart) Cells
AUTHORS: Moriah Beck, Abby Jurgensmeier

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

[STEPS]
?. Remove cells from the –80 °C freezer and place directly into your ice bucket.  Thaw on ice for 15 minutes.
?. Add DNA from plasmid to be transformed.  For ligations/PCR products use 10uL of ligation reaction per 50-100uL of cells.  For purified plasmid (miniprep), use 1 uL of plasmid per aliquot of cells.  Do not mix or stir the cells they are very fragile.
?. Incubate cells on ice with the DNA for 30 minutes, in the meantime make sure there is a 42°C water bath/block that has water in it.
?. Bring ice bucket with cells and a timer over to the water bath/heat block.  Immerse tube of cells in 42°C bath for 45-60 seconds.
?. Remove tube from waterbath and place directly on ice to recover for 2 minutes.
?. Add 900 uL of sterile LB (no antibiotics) and grow with shaking for 1 hour at 37 °C.
?. Plate out 50-200 uL on antibiotic selective plate of choice OR if cloning (small amounts of DNA for transformation) proceed to next step.
?. Spin cells in tabletop centrifuge for 1 minute at ~2,000 rpm, remove 700uL of media and resuspend the cell pellet in the remaining media in the tube.  Plate this onto the correct antibiotic LB plate.
TITLE: RCA of Circular Probe
AUTHORS: Chia-Hsien  Shih

[DESCRIPTION]
This protocol is to verify that circular probe can conduct RCA reaction.

[STEPS]
SECTION: Preparation
1. Dilute the 1µM Circular probe into 100nM
SECTION: Preparation
1.1. Add 5 µL of 1µM circular probe

SECTION: Preparation
1.2. Add 45 µL of RNase-free water

SECTION: Preparation
1.3. Spin down after vortex
SECTION: Protocol
2. Add 17.4 µL RNase-free water into a eppendorf

SECTION: Protocol
3. Add 3 µL of 10X phi29 polymerase reaction buffer

SECTION: Protocol
4. Add 3 µL of 100nM circular probe

SECTION: Protocol
5. Add 3 µL of 100nM miRNA

SECTION: Protocol
6. Add 3 µL of 2mM dNTPs

SECTION: Protocol
7. Add 0.6 µL of 10U/µl Phi29 polymerase

SECTION: Protocol
8. Pipetting the reaction solution
SECTION: Protocol
9. Incubate for 120 min at 31 Room temperature
SECTION: Protocol
10. Add 1.5 µL of 20X evagreen dye

SECTION: Protocol
11. Add 1.5 µL of 0.5M EDTA buffer to suspend the reaction

SECTION: Measuring
12. Load 20 µL of reaction solution into 384-well plate

SECTION: Measuring
13. Measure the fluorescence excitation and emission intensity
TITLE: Mouse OpenField + ANY-Maze Protocol
AUTHORS: Sélène Bonnet-Zahedi, Olivier George

[DESCRIPTION]
OpenField setup and analysis with ANY-Maze for the mouse room. September 2023

[GUIDELINES]
Try not to move the camera too much and not tare the grey tape on the floor. 

[STEPS]
SECTION: Setup and Record Experiment
1. - Plug the USB cord with orange tape connected to the ceiling camera to the power strip on the ground. This will turn on the camera
- Slowly and carefully move the apparatus to align with the grey tape on the floor

PLEASE BE CAREFUL NOT TO TARE THE GREY TAPE!

- Set the infrared lights around the apparatus.
- On the remote, press the infrared button (1)
- Switch off the lights
- Press the record button (2) on the remote to start recording the session (10 min recording)

 

SECTION: ANY-Maze Analysis
2. Transfer Data to Laptop:

- Turn on the laptop (Password: 2020).
- Plug the USB cord with orange tape to the laptop

PLEASE BE CAUTIOUS NOT TO MOVE THE CAMERA!

 
 
- This will open a new window on the laptop with the folder of the SD card.
- Transfer your files on the card into your personal folder. 
 
- Open the application "ANY-maze" on the desktop.
- Open the " Mouse OpenField 2023 Selene BZ protocol”.
SECTION: ANY-Maze Analysis
3. Protocol Configuration:

- Navigate to the "Protocol" tab.
- Under "Video source," choose "OG1 Camera" and select your video file to use.
- Apparatus Setup:  Define zone limits for each box:
           o Disabled unused chamber (if testing fewer animals).
           o Adjust orange rectangles for exterior and center zones. The center zone must always be 40% of the total area.
           o Also adjust the green ruler to match the apparatus side.
- Behavior Setup: Move to “Zones” and verify that the correct zones are highlighted in blue.
SECTION: ANY-Maze Analysis
4. Experiment Configuration:

- Move to the "Experiment" tab.
- Specify an experiment title.
- Add treatments with the corresponding number of animals (example: nicotine A, vehicle B)
- in the “View animals” tab, assign treatment to each animal (example = A for nicotine).
SECTION: ANY-Maze Analysis
5. Test Execution:

- Proceed to the "Test" tab.
- Ensure everything is set correctly.
- Click "Rewind and start the video at a test start" while pressing the start button.
- If the initial start doesn't work correctly, click "Stop," scroll down to the start button, and select "Start the video at a test start" twice to begin when desired.
- If issues arise, the experiment can be restarted by canceling in the left window.
- After completing one animal, load another video and repeat.
SECTION: ANY-Maze Analysis
6. Results Analysis:

- Go to the "Result" tab.
- Choose desired results, configure “Data Grouping”, adjust “Report Format,” and track plot.
- View Plots.
- Save what you want to save : protocol, text, track plot, ...
TITLE: Surgery free rat closed head repetitive mild injury model of traumatic brain injury 
AUTHORS: vedad.delic

[DESCRIPTION]
This is a surgery-free closed head weight drop injury protocol for repetitive mild TBI of adult rats. The model consists, of a guide tube and weight cylinders that deliver an injury to the calvarium of an anesthetized rat in prone position on a sponge causing rotational acceleration. 

Rats typically recover without incident with the most common adverse occurrence being a nose bleed. 4 injuries 2 weeks apart with 1.5 kg weight did not result in detectable pathology, but 8 injuries resulted in regional astrogliosis, microgliosis, encephalomalacia, without overt motor deficits. 

[GUIDELINES]
Injuries for this protocol were established in male Sprague-Dawley rats 10-12 weeks of age at the time of first injury and all injuries were spaced 10-14 days apart. 

It is highly recommended that investigators preform titrations for their specific application, rat strain, sex, and age, with injury ranges 0-1.75kg from 25 cm and 0.250 kg increments. 

[STEPS]
SECTION: Apparatus Setup
1. Ensure that the TBI platform is stable with pin in the 5th hole from the bottom
25 cm height.
SECTION: Apparatus Setup
2. Load weights. If loading multiple weights into the guide tube, load smaller weights first to ensure proper balance.
SECTION: Apparatus Setup
3. Turn on Isoflurane system with following settings:     

Open O2 gas tank connected to apparatus
Turn knob to 4-5% v/v for isoflurane
Gas delivery rate to 1.5 L/min for O2
Turn on valve for gas delivery to gas chamber and wait at least 10 seconds for chamber to fill before placing animal in


SECTION: Delivery of hit
4. Anesthetize the animal for 2 minutes in the gas chamber. Must be full anesthetized as to not respond to toe pinch.
Good time to weigh the rat

SECTION: Delivery of hit
5. Place anesthetized animal carefully into a decap cone and then under the weight-drop apparatus so that the top of the skull completely underneath the guide tube covering the entire calvarium. Arrange body in prone position on sponge. The guide tube should be lightly touching the head while the animal is on the sponge underneath. 

* Ensure that the rat forelimbs are folded back and that the tongue is not between incisors. 

SECTION: Delivery of hit
6. Pull the pin to release weights
SECTION: Delivery of hit
7. quickly retract weights up the guide tube using the attached string
SECTION: Delivery of hit
8. Place animal in recovery cage on its back, and time for the righting reflex
SECTION: Recovery observation
9. Full recovery
Quickening of breath
rat has righted itself completely with paws underneath he body
if minor bleeding present, it has rapidly resolved
Once fully recovered place rat in home cage
observe rats for 3 days, twice a day for continued signs of recovery

Not recovering
no breathing for more than 30s (rapid color change in tail, snout, paws)
very labored breathing that is not improving
seizures 
profuse bleeding
rat not woken up and not righted within 10 minutes
Euthanize rat