eamag/protocols-with-steps huggingface dataset:
TITLE: DNA extraction from mouthwash samples 
AUTHORS: Ahmed A Shibl, Anique Ahmad, Tsedenia Denekew, Mamon Abd AlBaqi, Aashish Jha

[DESCRIPTION]
DNA extraction

[BEFORE_START]
Set centrifuge to 4ºC
Keep CD2 solution on ice
Prepare and label collection tubes, microcentrifuge tubes, and MB spin columns

[STEPS]
1. Thaw mouthwash samples on ice for 30 min 

1.1. Transfer desired volume into 1.5 mL or 2 mL eppendorfs
1-2 mL 

2. Centrifuge transferred samples at maximum speed at 4 ºC for 10 min 
3. Discard the supernatant carefully without disturbing the pellet
4. Add 800 µL CD1 and vortex to resuspend pellet

5. Transfer entire eppendorf content to PowerBead Pro tubes
6. Secure PowerBead Pro tubes onto the bead beater and run at maximum speed for 5 min  

7. Centrifuge the PowerBead Pro tubes at maximum speed for 90 s 

8. Transfer the supernatant carefully without disturbing the pellet, into a clean 2 mL microcentrifuge tube
9. Add 200 µL CD2 into the 2 mL microcentrifuge tube and vortex for 10 s 

10. Centrifuge the 2 mL microcentrifuge tube at maximum speed for 90 s 
11. Transfer the supernatant carefully without disturbing the pellet, into a clean 2 mL microcentrifuge tube
12. Add 600 µL CD3 into the 2 mL microcentrifuge tube and vortex for 10 s 
13. Load 650 µL of the lysate onto MB spin columns and centrifuge at maximum speed for 90 s 
13.1. Discard flow-through and repeat step 13 to consume all the lysate
14. Place spin column onto new collection tube, add 500 µL EA, and centrifuge at maximum speed for 90 s 
15. Discard flow-through and place spin column back into the collection tube
16. Add 500 µL C5 onto spin columns and centrifuge at maximum speed for 90 s 
17. Discard flow-through and place spin column back into the collection tube
18. Centrifuge at maximum speed for 2 min and place spin column into 1.5 mL eppendorf (elution) tube
19. Add 50-100 µL nuclease-free water to the center of the spin column and leave at room temperature for around 5 min 
20. Centrifuge at maximum speed for 1 min, quantify using Qubit, flash freeze, and store at -20/80 ºC
3.1. Repeat step 1.1 if more volume is needed to see a pellet or a high yield of DNA is required
4.1. Spin down briefly
TITLE: Cryopreservation of Mammalian Cells (Suspension)
AUTHORS: Andrew Crowley

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A protocol for the preservation of suspension-type cells by freezing.</div><div class = "text-block">The protocol has been sucessfully used on:</div><div class = "text-block">    - THP-1 monocytes</div></div>

[STEPS]
?. Measure the density and viability of the cells
?. Gently pellet the cells via centrifugation
?. While centrifuging, use the live cell counts from step 1 to calculate the total volume required to resuspend the cells @
?. Gently aspirate the culture media from the pellet
?. Gently reuspend the cell pellet in cryopreservation media:    -  fetal bovine serum    -   cell culture media    -   DMSOUse the media volume calculated in step 3
?. Aliquot cells into labeled cryo vials
1 ml
?. Place cryo vials into a room temp Mr. Frosty freezing container
?. Freeze the container overnight
-80 °C
[(ie. overnight)]
?. Transfer the vials to liquid nitrogen for long term storage
?. Suggested cell culture media for step 5
TITLE: Energy solution preparation for OnePot PURE cell-free system
AUTHORS: Konstantinos Ragios

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">In this protocol we explain the procedure to create the Energy solution used for protein expression in OnePot PURE cell-free system. </div></div>

[STEPS]
?. For a 2.5x Energy Solution add  the materials needed tho a tube. The final concentration of the components is presented in Table 1.
Before adding each component make sure it is totally melted and then vortex for a few seconds
?. ABC1CompoundConcentrationUnits2Amino Acids0.75mM3Magnesium acetate29.5mM4Potassium glutamate250mM5DTT2.5mM6ATP5mM7GTP5mM8CTP2.5mM9UTP2.5mM10tRNA130UA260/mL11Creatine phosphate50mM12Folinic acid0.05mM13Spermidine5mM14HEPES125mM
ABC1CompoundConcentrationUnits2Amino Acids0.75mM3Magnesium acetate29.5mM4Potassium glutamate250mM5DTT2.5mM6ATP5mM7GTP5mM8CTP2.5mM9UTP2.5mM10tRNA130UA260/mL11Creatine phosphate50mM12Folinic acid0.05mM13Spermidine5mM14HEPES125mM
Because tRNA degrades over time before adding it to the solution you need to check the A260  units.
?. To complete the solution add biDistilled water until you reach the desirable volume, and then vortex for a few seconds
?. The Energy solution is stored at -80o C.
TITLE: BBB permeability and NDP-MSH PK study
AUTHORS: Pranay Srivastava

[DESCRIPTION]
This protocol is to measure the integrity of BBB and assess NDP-MSH concentrations in plasma and brain

[STEPS]
SECTION: BBB permeability
1. The integrity of BBB was measured through FITC-albumin (Millipore Sigma, Cat# A9771) leakage from vasculature into brain parenchyma as described previously (Chen et al. 2008). Mice were treated with MPTP+LPS and sacrificed after 6 h and 24 h after the last dose.
SECTION: BBB permeability
2.  Mice were anaesthetized by isoflurane and perfused intracardially with heparin (100 units/kg) followed by 5 ml FITC albumin at a concentration of 5 mg/ml in PBS with a flow rate of 1.5 ml/min.
SECTION: BBB permeability
3. Subsequently, the brain was isolated and incubated in 4% paraformaldehyde overnight. The solution was changed to 30% sucrose in PBS. Coronal sections of striatum were mounted and analyzed under fluorescence microscope (Olympus BX51 microscope). 
SECTION: NDP-MSH PK study
4. To assess NDP-MSH concentrations in plasma and brain, male mice were treated with
MPTP+LPS as described above and two concentrations of NDP-MSH (400 µg/kg and 1
mg/kg) and sacrificed after 5-, 30- and 90-min. 
SECTION: NDP-MSH PK study
5.  Blood samples were collected through cardiac puncture in 40 mM EDTA, and plasma was collected by centrifugation and stored at -80°C till further analysis. 
SECTION: NDP-MSH PK study
6. Whole brain was dissected and homogenized in PBS. 
SECTION: NDP-MSH PK study
7. Proteins in brain homogenate and plasma samples were crashed with 3 volumes of methanol containing internal standard (propranolol) and centrifuged. 
SECTION: NDP-MSH PK study
8.  Supernatants were analyzed by liquid chromatography/mass spectrometry (LC/MS). 

SECTION: NDP-MSH PK study
9. NDP-MSH in plasma and brain samples was detected by LC/MS through a service contract with Cyprotex, LLC, MA, USA.
TITLE: Collection of Protocols and Guidelines for Safety and Efficacy of Imatinib for Preserving Beta-cell Function in New-onset Type 1 Diabetes Mellitus
AUTHORS: Stephen.Gitelman , Jeffrey A. Bluestone

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a collection of protocols for: "Safety and Efficacy of Imatinib for Preserving Beta-cell Function in New-onset Type 1 Diabetes Mellitus".</div><div class = "text-block">This clinical study is supported by JDRF. The aim of the collection is to determine whether imatinib will slow the progression of the autoimmune destruction of ß cells and lead to the preservation of C-peptide secretion in T1DM and to assess Diabetes-related objectives and safety of Imatinib in new-onset type 1 diabetes mellitus".</div></div>

[STEPS]
TITLE: BW SARS-CoV-2 Laboratory Test
AUTHORS: ivan , peter.marx 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is the laboratory-based equivalent of the LAMP assay used for detecting SARS-CoV-2. A similar protocol is performed automatically within the Biology Works LLC at-home device.</div></div>

[STEPS]
?. [Assay]
Thaw reagents to room temperature
0 Room temperature
?. [Assay]
Vortex reagents
33
?. [Assay]
Briefly spin down components in a microcentrifuge
?. [Assay]
Prepare master mix in a 1.5mL tube with the following sub-steps
?. [Assay]
Add  to tube
12.5 µl
?. [Assay]
Vortex
33
?. [Assay]
Briefly spin down components in a microcentrifuge
?. [Assay]
Aliquot out 20uL into a PCR reaction plate
?. [Assay]
Dispense  of your sample solution into the well
5 µl
?. [Assay]
Seal plate with film
?. [Assay]
Centrifuge plate in plate centrifuge
Centrifuge: 500 1324
?. [Assay]
Place plate with reagents and sample into thermo-cycler
?. [Assay]
Run assay on thermo-cycler with the following protocol (specific to the ABI 7500): 1x 60x   and Record measurements
25 °C
64.9 °C
65 °C
?. [Assay]
Read results
?. [Assay]
Add    to tube
0.3125 µl
?. [Assay]
Add   to tube
4 µl
?. [Assay]
Add  to tube
0.5 µl
?. [Assay]
Add  to tube
2.8 µl
?. [Assay]
Close tube cap
TITLE: RNA extraction protocol for the Bungarus multicinctus by using TRlZOL reagent（Invitrogen）
AUTHORS: Boyang Liu, Liangyu Cui, Yue Ma, Diancheng Yang, Yanan Gong, Yanchun Xu, Shuhui Yang, Song Huang

[DESCRIPTION]
RNA was extracted from the muscle of a Bungarus multicinctus using the TRlzol reagent (Invitrogen, USA) and RNA extraction kit (Thermo Scientific, USA) in this protocol.

[STEPS]
SECTION: RNA extraction
1. The laboratory and test bench for RNA extraction were sterilized by alcohol and ultraviolet for 30 minutes. 
30 min 

All the equipment used in the experiment, such as mortar and pestle, were sterilized at high temperature one day before the experiment and dried in an oven. 

Masks and gloves should be worn throughout the RNA extraction process. Avoid exposure of RNases to experimental samples and cause RNA degradation.
SECTION: RNA extraction
2. On the clean bench, take 50 mg of scented glands and put them into a pre-cooled mortar to ensure that the liquid nitrogen has never passed the sample, quickly grind the sample into powder, add 1 ml TRIZOL, mix the tissue powder with TRIZOL, and put it into In a centrifuge tube, stand at room temperature for 15 min.
Room temperature 15 min 

SECTION: RNA extraction
3. Add 200 ml of chloroform, shake the centrifuge tube for 15 s to mix the chloroform and TRIZOL well.
Stand at room temperature for 10 min, put it in a refrigerated centrifuge and centrifuge at 4°C, 12000r/min, 15 min.
15 s 
Room temperature 10 min 
   

SECTION: RNA extraction
4. After centrifugation, the solution was separated into three phases. Take the upper aqueous phase into a new centrifuge tube, add three times the volume of ethanol to mix, stand at room temperature for 10 min.
Room temperature 10 min 

SECTION: RNA extraction
5. Add the mixed solution to the purification column, centrifuge at 12000r/min at 4°C for 2 min, repeat three times to ensure that ethanol is removed.
  

SECTION: RNA extraction
6. RNA was purified with RNA extraction kit (Thermo Scientific, USA), and RNA was eluted with DEPC.
SECTION: RNA extraction
7. Add RNase inhibitors to ensure that the RNA will not be degraded, and mark the outside of the centrifuge tube.

Check RNA purity and concentration, and confirm RNA integrity by electrophoresis.
TITLE: Striatal Cannula Window Implantation
AUTHORS: Shivathmihai Nagappan

[DESCRIPTION]
This protocol describes how to implant a cannula window over the dorsolateral striatum in mice to enable two-photon imaging of dopamine axons in the dorsal striatum.

[STEPS]
1. Prepare and administer appropriate pre-operative analgesics.
2. Anesthetize the mouse with isoflurane (5% in oxygen for induction and 1-2% in oxygen for maintenance) and secure in stereotaxic frame. Remove fur over the head and clean skin surface with Betadine solution. Then, make an incision over the midline, exposing the skull. Clip off excess skin around the skull.
3. Tilt the animal’s head 30 degrees horizontally clockwise or counterclockwise depending on which hemisphere the cannula will be implanted in. Perform a craniotomy, 2.75mm in diameter, using a circular hollow-core dental drill bit over the dorsolateral striatum (+0.3mm rostral, +2.7mm lateral to bregma).
4. Remove the skull piece and aspirate the underlying cortical tissue until the external capsule fibers over the striatum are exposed. To aspirate, apply gentle vacuum suction on the tissue while continuously washing with cold 1X PBS (Dombeck et al., 2010). Carefully remove some of the external capsule fibers until only a thin layer remains over the striatal surface.
5. Continue washing with cold 1X PBS until there is no bleeding into the cavity and the exposed striatal surface is clean.
6. Allow the surface to dry slightly and then apply a thin (~100-150μm) layer of Kwik-Sil over the exposed tissue. Insert the cannula (stainless-steel tube, 2.7mm outer diameter, 2.3mm inner diameter, 1.6mm length, covered at one end by a 1mm-thick glass coverslip that is glued on) into the cavity until the coverslip contacts the Kwik-Sil over the fibers. Allow Kwik-Sil to harden.
7. Attach a metal headplate to the skull surface, centered over the cannula, with dental cement (Metabond).
8. Once the Metabond hardens, remove the mouse from anesthesia and place it back into its home-cage. Allow it to recover over a heating pad.
9. Check the animal’s recovery 24 hours post-surgery and readminister appropriate analgesics.
TITLE: FCMPASS - Light scatter calibration
AUTHORS: Joshua Welsh, Jennifer Jones

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol outlines the steps required to input light scatter calibration parameters using the FCMPASS software. This is one of a number of protocols in the pipeline for performing small particle calibration using the fcmpass software package.</div></div>

[STEPS]
?. If light scatter calibration is being performed click the '+' button to add a calibration parameter to the table. If light scatter calibration is not required, click 'Next'.
?. If you have not yet defined the light scatter bead sets in Catalogue', click 'Catalogue' and complete as outlined in the protocol.
?. Double click the ‘Scatter Parameter’ field to change which parameter is being used for light scatter calibration.
?. Alter the ‘Scatter Wavelength (nm)’ to the relevant wavelength for the parameter being used to calibrate light scatter.
You will see that the ‘Sheath RI’ field will automatically update when this is altered. In the background reference bead, core-shell model, and homogenous sphere model refractive indices will all also be updated.
?. If the selected ‘Scatter Parameter’ was used as a triggering threshold then the ‘Scatter Threshold’ field will automatically update to show the values used as thresholds in the .fcs files loaded. Select a ‘Scatter Threshold’ by double clicking the field and selecting and option from the dropdown menu. A custom entry can also be inputted.
?. Load the light scatter reference beads used by double clicking the ‘Bead Set’ field. Once loaded the beads within the set will populate the bottom table.
?. The ‘Sheath RI’ field automatically accounts for ‘Scatter Wavelength’ but can be updated manually by double clicking the field.
?. In the bottom table enter the median scatter parameter statistic for each population. The acquired CV can optionally also be completed, its use will, however, only be used for plotting purposes and not alter the model calculations.
?. Once complete click ‘Next’.
Custom core-shell models, solid sphere models, plot data points, modelling parameters, and output settings can be entered or altered by clicking the ‘Advanced Settings’ button. By default, three EV core-shell models relating to high, medium, and low EV refractive indices are calculated. All core-shell models assume a 5 nm shell thickness.
TITLE: Phonological Short Term Memory tasks for Italian-English comparison
AUTHORS: Chiara Valeria Marinelli, Pierluigi Zoccolotti, Cristina Romani

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:center"><span style = "font-weight:bold;">Phonological Short Term Memory tasks for Italian-English comparison</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:justify"></div><style>
							  .justify:after {
							    content: "";
							    display:inline-block;
							    width: 100%;
							  }
							</style></div><div class = "text-block">An attempt was made to develop tasks that would be comparable between the Italian (i.e., a consistent orthography) and English (i.e., an inconsistent orthography)versions. </div><div class = "text-block"><span style = "font-style:italic;">Phonological STM </span><span>was measured by a non-word repetition and a digit span tasks. </span></div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[GUIDELINES]
Example of two PCR'd gDNA extracts, one from a nucleofected cell and one from an unzapped control, saw heteroduplexing in the edited sample and tried to get rid of it by reconditioning and/or adding more polymerase.<br /><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/4cc75n.jpg" alt="" data-src="https://s3.amazonaws.com/pr-journal/4cc75n.jpg" data-ofn="pic1.jpg" /><br /><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/4ec75n.jpg" alt="" data-src="https://s3.amazonaws.com/pr-journal/4dc75n.jpg" data-ofn="pic2.jpg" />

[STEPS]
?. 
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?. 
?. 
?. 
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?. 
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?. 
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?.
TITLE: Scam Alert:- Clinical CBD Gummies &#34;Shark Tank&#34; Full Data
AUTHORS: jimmyrobert  

[DESCRIPTION]
At this point you need just to avoid searching and confusing your mind. Just focus using one product, rely on it, and then step into the planet of healing and painless fun. From the comfort of home so you will be purchasing the very best product called  Clinical CBD Gummies  that too at the very best price what else could you ask for. Early bird offers are limited and grab prior to others do.

[STEPS]
TITLE: LRRK2 PhosphoSens Assay
AUTHORS: Nicolai D. Raig, Stefan Knapp

[DESCRIPTION]
With this enzymatic activity assay we were able to determine IC50 values of published inhibitors aswell as newly synthesized compounds that inhibit LRRK2. The assay is based on the in vitro phosphorylation reaction between the enzymatic active LRRK2(RCKW)-construct and the SOX-based substrate peptide (AQT0615 from AssayQuant Technologies).

[STEPS]
2. Pipett a dilution series of eleven concentrations between 15µM and 0.4nM (calculated with an assay volume of 10µL) of the compounds into white 384-well plates (Greiner 781207) as duplicates with an ECHO acoustic dispenser (Labcyte). Pipett a equivalent of DMSO in two wells per compound as 0% (without protein and compound) and 100%(without compound) control.
3. Dilute the purified LRRK2RCKW to 22nM with the reaction buffer (50mM HEPES buffer (pH7.5), 10mM MgCl2, 1% glycerol, 1mM DTT, 0.2mg/mL BSA, 0.01% Tween20, 5µM AQT0615) and add 10µL to each well, except the 0% control wells,  with a multichannel pipette (Eppendorf). Add   10µL of pure reaction buffer to the 0% control wells.
1. Make 10mM, 100µM and 1.6µM stock solutions of the compounds in DMSO.
4. Pipett 5nL 100mM ATP stock into each well with the ECHO acoustic dispenser (Labcyte).
5. Centrifuge the plates at 1500gn for 2min, before incubating the reaction at room temperature for 4h.
6. Measure the fluorescence after 360nm excitation at 487nm emission with a PHERAstar plate reader (BMG Labtech).
7. Normalize the response to the average 0% and 100% controls. Calculate the IC50 values via non-linear regression of the log[inhibitor] vs. the normalized response with GraphPad Prism 8.
TITLE: Enzymatic Assay of Protease Using Azocasein as Substrate
AUTHORS: Neilier Junior

[STEPS]
?. [Reagent Preparation]
100 mM Tris-HCl buffer, pH 8.0, 20 mM CaCl2, at 37 °C.2.0% (w/v) Azocasein SolutionHeat gently (do not boil) to 50 - 60 °C for 10 min with stirring.Adjust the pH to 8.0 at 37 °C, if necessary, with either 1.0 M NaOH or 1.0 M HCl.110 mM Trichloroacetic Acid Reagent (TCA). Dilute with deionized water.500 mM Sodium Hydroxide (NaOH) Solution. Prepare in deionized water.2.0% (w/v) Azocasein SolutionHeat gently (do not boil) to 50 - 60 °C for 10 min with stirring.Adjust the pH to 8.0 at 37 °C, if necessary, with either 1.0 M NaOH or 1.0 M HCl.110 mM Trichloroacetic Acid Reagent (TCA). Dilute with deionized water.500 mM Sodium Hydroxide (NaOH) Solution. Prepare in deionized water.Heat gently (do not boil) to 50 - 60 °C for 10 min with stirring.Adjust the pH to 8.0 at 37 °C, if necessary, with either 1.0 M NaOH or 1.0 M HCl.110 mM Trichloroacetic Acid Reagent (TCA). Dilute with deionized water.500 mM Sodium Hydroxide (NaOH) Solution. Prepare in deionized water.Check how many samples will be analyzed to calculate the required volume of each solution to be prepared.
?. [Procedure]
Pipette (in microliters) the following reagents into 2.0 mL microtubes.  ABC1BlankTest2Tris-HCl buffer750 μL450 μL3Azocasein750 μL750 μL4Mix and equilibrate to the at desired temperature. Then add:*5Sample (enzyme source)-300 μL6Mix and incubate at desired temperature for exactly 30 min.*7Remove a 1 mL aliquot from both (test and blank) solutions and place into 2.0 mL microtubes. Then add:8TCA1000 μL1000 μL9Centrifuge at 20,000 g for 10 min. Remove a 1 mL aliquot from supernatant (test and blank) and place into 2.0 mL microtubes. Then add:*10NaOH1000 μL1000 μL11Mix and transfer the Test and Blank solutions to suitable cuvettes. Measure the A440nm for Test and Blank using a spectrophotometer.*
ABC1BlankTest2Tris-HCl buffer750 μL450 μL3Azocasein750 μL750 μL4Mix and equilibrate to the at desired temperature. Then add:*5Sample (enzyme source)-300 μL6Mix and incubate at desired temperature for exactly 30 min.*7Remove a 1 mL aliquot from both (test and blank) solutions and place into 2.0 mL microtubes. Then add:8TCA1000 μL1000 μL9Centrifuge at 20,000 g for 10 min. Remove a 1 mL aliquot from supernatant (test and blank) and place into 2.0 mL microtubes. Then add:*10NaOH1000 μL1000 μL11Mix and transfer the Test and Blank solutions to suitable cuvettes. Measure the A440nm for Test and Blank using a spectrophotometer.*
?. [Calculation]
ΔA440nm = A440nmTest - A440nmBlank
TITLE: Lemnaceae (Duckweed) culturing protocol
AUTHORS: o.pogoutse, Jason Laurich, Chris Carlson

[DESCRIPTION]
Protocol for culturing duckweed. 

[STEPS]
SECTION: YMA Media Preparation
2. To prepare 500ml of 1x YMA media in the biosafety cabinet  pour 100 ml of sterile
5x YMA media into sterile cylinder.
SECTION: Sterilisation and Setup
1. Spray down inside of biosafety cabinet with ethanol, including materials
 to be used.
SECTION: YMA Media Preparation
3. Next add sterile H2O till the beaker contains around 350 ml.
SECTION: YMA Media Preparation
4. Next add 75 ml of CaCl2 stock solution (sterile stock 1.5mg/ml).
SECTION: YMA Media Preparation
5. Top off  with sterile H2O to 500 ml total volume. 
SECTION: YMA Media Preparation
6. Add 500 μL of 1000x stock solution of MgSO4 x 7H2O (150mg/ml, filter sterilized) to media. Transfer prepared media to a sterile bottle and mix by inverting the bottle a few times.
TITLE: Tris Buffered Saline (TNT)
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used to prepare Tris Buffered Saline (TNT). 10X TNT is the stock solution used to prepare 1X TNT, which equilibrates tissue and maintains pH in physiological range.</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]