eamag/protocols-with-steps huggingface dataset:
TITLE: Seeding 
AUTHORS: Francisco Javier Quero, Alfredo L´Homme Iriarte, Laura Armero

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes the procedures to seed the </span><span style = "font-style:italic;">Bacullis subtilis </span><span>inside the microfluidic chip to create the biofilms.</span></div></div>

[STEPS]
?. [Day before experiment]
Cellls from stock were streaked onto LB agar plate and incubated at 37 ºC overnight.
?. [Day of the experiment]
A single colony was picked from the plate and inoculated into 3 ml of LB broth in a 50 ml conical tube, and then incubated at 37 uC in a shaker.
?. [Day of the experiment]
After 2.5 h of incubation, the cell culture was centrifuged at a relative centrifugal force of 2,100 for 1 min.
?. [Day of the experiment]
The cell pellet was re-suspended in MSgg and then immediately loaded into microfluidics.
?. [Day of the experiment]
After the loading, cells in the microfluidic chamber were incubated at 37 ºC for 90 min, and then the temperature was kept at 30 ºC for the rest of the experiment.
TITLE: Microplate reader operating procedure
AUTHORS: Shuning   Guo, Zhujun Wei

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Use Tecan Spark® multimode microplate reader to measure absorbance or fluorescence intensity of green fluorescence protein.</div><br/></div>

[STEPS]
?. Turn on the power and warm up for 10 minutes.
?. Open the program and select "New" to create a new process.
?. Select "Absorbance" in the left column. There is a schematic of a 96-well plate in the middle of the screen.
?. You can select the hole to be used according to the spotting hole, and the selected hole will turn blue.
?. Put in the 96-well plate, set the parameters, click "start" to measure the absorbance, and then remove the 96-well plate.
?. Wait a moment, it will generate an Excel file with results and various parameters. Save and copy the file to the u disk and process the data.
TITLE: Reverse transcription of RNA to cDNA
AUTHORS: iGEM Dusseldorf

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Synthesizing cDNA (for qPCR)</div></div>

[STEPS]
?. [Preparation of 1.33X yellow buffer]
33,25 µL 40x yellow sample buffer + 966,75 µL H2Ooptional: This was used to be able to see the pipetting scheme for qPCR more easily
?. [Start]
In PCR stripes, pipet in the following order:100 ng template RNA1 µL random hexamer primersRNase-free H2O to 13 µL volume
?. [Start]
Prepare a Mastermix of the following reagents:4 µL 5x RT-buffer2 µL dNTP Mix (10 mM of each nucleotide)1 µL RevertAid Reverse Transcriptase
?. [Start]
Add 7 µL Mastermix to each reaction
?. [Start]
For your -RT control, pipet 100 ng RNA in PCR stripes, leave out buffer and everything, just add H2O to a final volume of 20 µL. Mark as -RT so you can distinguish it from your actual cDNA!
?. [PCR]
PCR protocol:  AB110 min25 ˚C260 min42 ˚C310 min70 ˚C
AB110 min25 ˚C260 min42 ˚C310 min70 ˚C
?. [PCR]
Add 60 µL 1,33 x yellow sample buffer to each reactionoptional: This was used to be able to see the pipetting scheme for qPCR more easily
?. [PCR]
cDNA can be stored at 4 ˚C for a short time, otherwise, freeze at -20 ˚C
TITLE: Endogenous coimmunoprecipitation
AUTHORS: michela.deleidi, Pascale Baden

[DESCRIPTION]
Protocol used for immunoprecipitation of HSP60 and LONP1 in HEK cells to show the interaction with V5-Flag-tagged WT-GCase

[STEPS]
7. Spin cells in a centrifuge at 250g for 5 minutes at room temperature.
8.  Remove the supernatant and wash cells in PBS.
9. Repeat steps 7 and 8 for a total of 2 washes.
10. Lyse cells in 1% TBS + 0.5% NP40 + PI/PHI (Pierce #A32959)
SECTION: Endogenous coimmunoprecipitation
1. Wash Protein G agarose fast-flow beads in TBS 1X + 0.05% NP40.To this
end, the beads were incubated with lysis buffer on the spinning wheel (25 RPM) for 2
–5 min at 4 °C, followed by a 1 min centrifugation at 500 RPM 4 °C.
SECTION: Endogenous coimmunoprecipitation
2. Repeat step 1 for a total of 3 washes.
SECTION: Endogenous coimmunoprecipitation
3. Incubate 20ul of pure beads with 5ug of anti-LONP1 antibody or 5ug of normal rabbit IgG as control (or 3ug of anti-Hsp60 and 3ug of mouse IgG as control) and add 300ul of washing buffer to each tube. 
SECTION: Endogenous coimmunoprecipitation
4. Incubate 2h at 20RPM on a rotating wheel at 4°C
11. After 2h incubation, wash beads again 3 times in washing buffer.
6. Detach cells using Accutase for 5 minutes at 37°C and collect them.
5. Meanwhile, detach HEK cells using Accutase for 5 minutes at 37°C and collect them.
12. Incubate antibody-coated beads with 3.7 ug of lysate on the spinning wheel for 2h.
13. After 2h incubation, wash beads again 3 times in washing buffer.
14. Elute by boiling the beads twice with 2x Laemmli buffer at 95°C for 8min at 300rpm in a thermoblock.
15. Spin beads for 1 minute at 10000 RCF.
16. Collect supernatant and proceed with western blot analysis.
TITLE: PCR mixture and condition (2X SUPERGREEN PCR MASTER MIX)
AUTHORS: Yin-Tse  Huang

[DESCRIPTION]
PCR mixture and condition (2X SUPERGREEN PCR MASTER MIX)

[STEPS]
SECTION: For generic primers
1. PCR mixture PCR condition for generic primers
 

SECTION: For barcoded primers
3. PCR mixture for barcoded primers

 

SECTION: For generic primers
2. PCR condition for generic primers

 

SECTION: For barcoded primers
4. PCR condition for barcoded primers
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This tutorial explains how to download entire libraries of data from the VIROME web application. <br /><br />Wommack, K. E., J. Bhavsar, S. W. Polson, J. Chen, M. Dumas, S. Srinivasiah, M. Furman, S. Jamindar, and D. J. Nasko. 2012. VIROME: a standard operating procedure for analysis of viral metagenome sequences. Standards in Genomic Sciences 6:427-439 [<a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3558967/" target="_blank">PMC3558967</a>] <br /><br /><a href="http://virome.dbi.udel.edu" target="_blank">See site</a> for more information.

[GUIDELINES]
See full video below for more information.<br /><br /><iframe src="https://www.youtube.com/embed/sVoku4GyK6s" width="420" height="315" frameborder="0" allowfullscreen="allowfullscreen"></iframe><br /><br />YouTube link: <a href="https://www.youtube.com/watch?v=sVoku4GyK6s" target="_blank">https://www.youtube.com/watch?v=sVoku4GyK6s</a>

[STEPS]
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TITLE: Preparation of Chemically Competent Cells
AUTHORS: NUS iGEM

[STEPS]
?. Transfer   of overnight culture into   LB in a flask
1 ml
50 ml
?. Incubate at   at   until OD600 = 0.6
37 °C
Centrifuge: 225 33
?. Transfer culture to   falcon tube
50 ml
?. Incubate culture on ice for
?. Centrifuge tube at  ,   for
4 °C
Centrifuge: 5000 33
?. Discard supernatant and resuspend pellet in   of   magnesium chloride solution
30 ml
?. Centrifuge tube at  ,   for
4 °C
Centrifuge: 5000 33
?. Discard supernatant and resuspend pellet in   of  calcium chloride solution
20 ml
?. Incubate sample on ice for
?. Centrifuge tube at  ,   for
4 °C
Centrifuge: 5000 33
?. Resuspend pellet in   of cold mixture comprising 20% glycerol and 80%   calcium chloride solution
1.5 ml
?. Aliquot   of mixture into 1.5mL eppendorf tubes
60 µl
?. Store competent cells in
-80 °C
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Cellectis, a French biopharmaceutical company, recently published a paper in the Scientific Reports, a publication of the Nature Publishing Group, and reported on the details of the CubiCAR developed by the company. CubiCAR is a novel chimeric antigen receptor (CAR) that carries a multifunctional tag for purification, detection, and depletion of CAR-T cells. These new features are expected to simplify the manufacturing process of CAR-T cells and improve the effectiveness of tracing and depleting CAR-T cells in the clinical environment.</p>

[STEPS]
?.
TITLE: Arduino
AUTHORS: bibewih  

[DESCRIPTION]
This guide shows how to assemble The electronic components of the Mock Coastal Environment. This build uses a prototyping breadboard and wires. It should be noted that soldering of components and wires would make a more permanent solution but is outside the scope of this protocol.

[STEPS]
1. Wire components to Arduino board as depicted in wiring diagrams.
1.1. Setup breadboard by attaching a red wire from (+) to 5V on  and black (-) wires to GND on Arduino board. 
1.2.   
Wire the waterproof thermometer sensor as shown in the diagram above. The red wire to (+) and the black wire to (-) on breadboard. A 4.7k Ohm resistor links the red wire to the yellow (depicted blue in diagram) wire. The yellow wire is also connected to the 7 digital pin on the Arduino (teal lead on diagram). Thread the sensor through the hole in the metal collar of the water bath with enough wire to submerge in water.

2. Download Arduino IDE from https://www.arduino.cc/en/software on your personal computer.
3. Open Arduino IDE and connect Arduino board to computer with USB cable.
4. Obtain code for Arduino at https://anonymous.4open.science/r/Mock-Coastal-Environment-8C1E/
5. Upload code to Arduino. Follow the guide here:
https://docs.arduino.cc/software/ide-v2/tutorials/getting-started/ide-v2-uploading-a-sketch
1.3. Connect LCD screen by connecting SDA and SCL to respective digital pins on the Arduino. The VCC should be connected to positive (+) and GND to negative (-) on the breadboard.
1.4. Connect IOT relay to Arduino by connecting the 6 digital pin on the Arduino to the positive (+) terminal on the relay and  connect the negative (-) terminal on the relay to negative (-) on the breadboard.
1.5. Cut 8" from the white wire of both 3d printer heating pads used to assemble the water bath. Attach fork spade wire connectors to both wires. use these connectors to connect both to the V+ screw terminal on the power supply located in the electronic enclosure on the water bath. Connect the other ends of the white wires to two of the relays on the 4ch relay board. connect the white wires on the heating pads to the other connection on the relay. Attach fork spade wire connectors to the unconnected wires on the heating pads and attach them to the V- screw terminal on the water bath's power supply. Connect VCC and GND on 4ch relay board to positive (+) and (-) on breadboard respectively. Connect the inputs for the used relays to digital pins 8 and 9 on the Arduino.
6. If using Raspberry Pi, setup using the instructions provided here: https://projects.raspberrypi.org/en/projects/raspberry-pi-setting-up
7. Obtain Temperature scripts from same link in step 5 and load them on the computer or raspberry pi.
8. Turn on water bath, IOT relay, and Arduino. Connect computer or raspberry pi to Arduino using USB cable. 
9. Run Temperature script on computer or raspberry pi.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Preparation of trace metal stock solutions for cyanobacteria trace metal mixture (CTMM)

[BEFORE_START]
Before culturing cyanobacteria, read the publication by L.R. Moore et. al. (2007) Limnol. Oceanogr. 5: 353-362.<br />Purchase the highest quality chemicals to avoid trace metal contamination. <br /> Acid-wash all re-uesable containers.

[GUIDELINES]
Prepare stock trace metal solutions using the amounts below for 100 mL or (50 mL) volumes:<br /><br />2.30g (1.15g) ZnSO<sub>4</sub>·7H<sub>2</sub>O <br />1.19g (0.595g) CoCl<sub>2</sub>·6H<sub>2</sub>O <br />17.81g (8.905g) MnCl<sub>2</sub>·4H<sub>2</sub>O <br />0.726g (0.636g) Na<sub>2</sub>MoO4·2H<sub>2</sub>O <br />1.73g (0.865g) Na<sub>2</sub>SeO<sub>3</sub> <br />2.38g (1.19g) NiCl<sub>2</sub>·6H<sub>2</sub>O

[STEPS]
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TITLE: Colony PCR
AUTHORS: An.Huang

[DESCRIPTION]
Colony PCR using the whole organism of bacteria instead of purified DNA template. This simplifies PCR procedure. This protocol helps conduct a simple colony PCR procedure.

[STEPS]
SECTION: Colony PCR
7. Add 30 µL TE buffer to X+2 1.5ml microcentrifuge tubes each. Label the tubes as "1, 2, 3, ..., X, +, -".
 

SECTION: Colony PCR
9. Place the tubes in a heating block, heating at 100 °C for 5 min.

SECTION: Colony PCR
10. Prepare Master Mix for colony PCR. The recipe for the Master Mix is as follows:
    
 




SECTION: Colony PCR
12. Pipette 15 µL from the Master Mix into all X+2 0.2 mL PCR tubes.
SECTION: Colony PCR
14. Place the X+2 PCR tubes in Thermocycler. PCR procedure will be set as the following programme:

1 cycle of 95 °C 
30 cycles of 94 °C 
                       50 °C 
                       72 °C 
Final extension 72 °C 

SECTION: Preparation for experiment
6. Prepare several sterilized 1.5ml microcentrifuge tubes. 
 

SECTION: Preparation for experiment
1. Pick several colonies of bacteria from the plate using pipette tips.
SECTION: Preparation for experiment
2. Put the selected colonies into different 15mL centrifuge tubes, each with 5 mLLB broth.

SECTION: Preparation for experiment
3. Incubate in an orbital shaker at   overnight.


SECTION: Preparation for experiment
4. Pipette 30 µL culture from each tube to different agar plates. Spread the culture evenly on the plate.

SECTION: Preparation for experiment
5. Incubate the plates in a biochemical incubator at 37 °C overnight.

 


SECTION: Colony PCR
8. Pick one colony from each plate 。 using a sterilized pipette tip and put the colonies into different 1.5ml microcentrifuge tubes numbered "1, 2, 3, ..., X"

SECTION: Colony PCR
13. Pipette 5 µL from the colony lysate from tube  "1, 2, 3, ..., X" into corresponding 0.2 mL PCR tubes.
Add 5 µL plasmid into "+" PCR tube. Add 5 µL ddH2O into "-" PCR tube.
 

SECTION: Colony PCR
11. Label X+2 0.2mL PCR tubes as "1, 2, 3, ..., X, +, -"
SECTION: Colony PCR
15. When the programme is finished, store the tubes at 4 °C
TITLE: Q5 PCR DNA Amplificaiton (Protocol for Q5® High-Fidelity 2X Master Mix)
AUTHORS: Alba Balletbó

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is for PCR with Q5® High-Fidelity 2X Master Mix</div></div>

[STEPS]
?. Gently mix the PCR reactions and transfer the tubes to a thermocycler. Thermocycling conditions for a routine PCR:  ABC1StepTemperatureTime2Initial Denaturation98°C30 seconds325–35 Cycles98°C5–10 seconds4*50–72°C10–30 seconds572°C20–30 seconds/kb6Final Extension72°C2 minutes7Hold4–10°C&nbsp;
ABC1StepTemperatureTime2Initial Denaturation98°C30 seconds325–35 Cycles98°C5–10 seconds4*50–72°C10–30 seconds572°C20–30 seconds/kb6Final Extension72°C2 minutes7Hold4–10°C&nbsp;
?. Add all components in a 250 μL tube making up to a 25 or 50 μl reaction. If performing various PCR with different templates, a Master Mix is recommended to be done.
When doing a Master Mix always add Q5 enzyme last, then vortex the solution briefly and centrifugate before use. 
'Pro-Tip': Use DMSO 3% or 5X GC enhancer as PCR additives when amplifying particularly difficult or high GC amplicons.
TITLE: U Mass - Glucose Tolerance Test with insulin secretion
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">Glucose tolerance test with insulin secretion measures systemic clearance of glucose and systemic appearance of insulin following an intraperitoneal bolus injection of 20% dextrose. This experiment measures insulin sensitivity and insulin secretion (pancreatic -cell function) in awake mice assuming that there are no alterations in systemic insulin clearance.  Insulin sensitivity and pancreatic -cell function are altered in obese mice.</div></div>

[STEPS]
?. Mice may be fasted overnight (~15 hours) or for 5 hours prior to the start of experiment.
?. Collect plasma sample (20 l) before the start of experiment (basal-0 min) to measure basal glucose and insulin levels.
?. Administer intraperitoneal injection of 20% dextrose (1 or 2 g/kg body weight) using an insulin syringe.
?. Collect plasma samples (20 l) at 10, 20, 30, 60, 90, and 120 min following injection to measure circulating glucose and insulin concentrations.
?. For data analysis, plasma glucose levels vs. time after injection are plotted, and areaunder-curve may be calculated to estimate insulin sensitivity. Also, plasma insulin levels vs. time after injection are plotted, and area-under-curve may be calculated to estimate insulin secretion (pancreatic -cell function).
TITLE: 07 Extraction of Plasmid
AUTHORS: TJUSLS China

[STEPS]
?. Collect the E. coli solution into the EP tube. Centrifuge at 12,000 rpm in a rotor for 1 minute. Remove the clear supernatant liquid.
?. Add 250μL P1 (RNase A added, kept at 4 °C)to the EP tube to suspend bacterial precipitation.
4 °C
250 µl
?. Add 250μL P2 to the EP tube, shake slightly up and down 6-8 times to lyse bacteria.
250 µl
?. Add 350μL P3 and invert the tube immediately and gently 6-8 times. Then centrifuge it at 12000rpm, 25℃ for 10 minutes.
350 µl
25 °C
?. Regenerate column CP3 while centrifugation. Add 500μl Buffer BL. Centrifuge for 1 min at 12,000 rpm. Discard the flow-through.
500 µl
?. Move the clear supernatant liquid to CP3, at 12000rpm, 25℃ centrifuge for 30 seconds.
25 °C
?. Add 600μL PW to adsorption column CP3, 12000rpm, 25℃ centrifuge for 30 seconds.
25 °C
?. Repeat step 7.
25 °C
?. Move the adsorption column CP3 to new clean centrifuge tubes and then keep them opening for 5 minutes, so that the ethanol in the PW can be sufficiently volatilized.
?. Drop 50μL 65°C ddwater into the middle of the adsorption membrane, static for 2min. Then centrifuge for 2 min at 12,000 rpm to collect DNA solution in EP tube.
50 µl
65 °C
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<span style="text-decoration: underline;"><strong>References:</strong></span><br /> <span style="font-family: Arial,Helvetica,sans-serif;">Becton Dickinson Immunocytometry Systems Source Book (1989) 2.10 <br /></span> <span style="font-family: Arial,Helvetica,sans-serif;">Lanier, L.L., and Warner, N.L. (1981) Paraformaldehyde Fixation of Hematopoietic Cells for Quantitative Flow Cytometry (FACS) Analysis. Journal of Immunological Methods 47, 25</span> <br />C.A. Williams, M.W. Chase, Stabilizes cell membranes and preserves cell morphology. <em>Methods Immunol. Immunochem.</em> New York <strong>5</strong>, (1976)

[GUIDELINES]
Needed:<br />
<ul style="list-style-type: undefined;">
<li><a href="http://www.canemco.com/product-catalog/chemicals/18-products/96-fixatives#16%_Formaldehyde_Solution" target="_blank">16% Formaldehyde </a></li>
<li>PBS</li>
<li>Ice</li>
</ul>
<span style="text-decoration: underline;"><strong>Working Dilutions:</strong> </span><br /> <span style="font-family: Arial,Helvetica,sans-serif;">Thawed aliquots are stable at 4&ordm; C for up to 2 weeks.<br /></span><span style="font-family: Arial,Helvetica,sans-serif;">Dilute 1 part 2% PFA to 3 parts cells in PBS (eg 60 &micro;l + 180 &micro;l to yield 0.5% final concentration).</span>

[STEPS]
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