eamag/protocols-with-steps huggingface dataset:
TITLE: PCR
AUTHORS: Zhujun Wei

[STEPS]
?. Add the following reagent to a PCR tube.(50 μl).  AB12×high Taq Master Mix (Enzyme)25 μl2Template2 μl3Forward Primer (10 μM)2 μl4Reverse Primer (10 μM)2 μl5ddH2O19 μl
AB12×high Taq Master Mix (Enzyme)25 μl2Template2 μl3Forward Primer (10 μM)2 μl4Reverse Primer (10 μM)2 μl5ddH2O19 μl
?. Program the thermocycler as follows:  AB1TemperatureTime298℃5 min398℃15 s4Tm-4℃15 s572℃(5-10s)× (1 kb)672℃5 min74℃∞
AB1TemperatureTime298℃5 min398℃15 s4Tm-4℃15 s572℃(5-10s)× (1 kb)672℃5 min74℃∞
TITLE: Total Starch Enzymatic Digestion 
AUTHORS: Lynn    Doran, Amanda  P. De Souza

[DESCRIPTION]
Enzymatic digestion of total soluble starch to glucose in plant tissue extracts for preparation for quantification via the GOD-POD Method (NZYtech).

[BEFORE_START]
Extract and dry total starch pellet from plant tissue per Extraction of Non-Structural Carbohydrates (Total Soluble Sugars + Starch) in Plant Tissues.

[STEPS]
1. Prepare fresh daily 120 U/mL α-amylase in MOPS buffer. 1 mL per sample will be needed. Initial concentration of α-amylase is 1000 U/mL.  Use C1V1 = C2V2 to calculate the volume of α-amylase and MOPS buffer to use.
 

2. Prepare fresh daily 30 U/mL amyloglucosidase in acetate buffer. 1 mL per sample will be needed. Initial concentration of amyloglucosidase is 3260 U/mL.  Use C1V1 = C2V2 to calculate the volume of amyloglucosidase  and acetate buffer to use.
 

3. Heat the water bath to 75 °C.

4. Add 500 µL of α-amylase in MOPS buffer (120 U/mL) to each sample tube.

6. Incubate for 30 min at 75 °C in the water bath.

5. Vortex to suspend all solids.  Flicking the tube may help dislodge the dried starch pellet.
7. Add another 500 µL of α-amylase in MOPS buffer (120 U/mL) to each sample tube.
8. Vortex to suspend all solids.
9. Incubate for 30 min at 75 °C in the water bath.

10. Cool down the water bath to 50 °C, tubes can be stored at room temp on the counter while water bath cools.
 

11. Add 500 µL amyloglucosidase in acetate buffer (30 U/mL) to each sample tube.

13. Incubate for 30 min at 50 °C in the water bath.

12. Vortex to suspend all solids.
14. Add another 500 µL amyloglucosidase in acetate buffer (30 U/mL) to each sample tube.

15. Vortex to suspend all solids.
16. Place the tubes in ice to stop the reaction, until cool to the touch.
17. Proceed to total starch (as glucose) quantification by NZYtech GOD-POD method or store the samples at -20°C up to one month.
TITLE: Conducting Online Research With Infants
AUTHORS: ajthomas 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a step-by-step process for running synchronous online studies with infants: studies in which you meet parents and infants via video chat. This method is meant to be very similar to methods commonly used in infant labs. We use Zoom (to meet with parents) and youtube for displaying videos, but there are other options (e.g., Slides.com for displaying videos). This method does not employ any habituation techniques or infant-controlled stimulus presentation: All participants see the same number of familiarization videos and test videos. The test videos are also a set length because we measure where on the screen the infants are looking. </div></div>

[STEPS]
?. [Stimuli Set Up]
Make videos of stimuli by using keynote or powerpoint. Once you have the stimuli you want to use. You can use keynote or powerpoint to make a long video that includes attention getters etc. Make your keynote file that includes videos and attention getters in the order you want them. Then select File->Export->Export to movie. If you don't want time between slides, make sure you set the before and after cells to zero. This will give you videos of the stimuli you can use in your experiment. Double-check that timing etc. is correct in the exported video. If you have several versions of your stimuli for counterbalancing etc, name them 'A', 'B' or '1', '2'.
?. [Stimuli Set Up]
Make introduction/wrap-up videos. Slides that can be used for these videos can be found here:  and here:  These videos should include the following:
?. [Stimuli Set Up]
Intro Slide: this is the first slide that parents will see and should have the approximate duration of the study, one sentence about what the study is about/what to expect, a reminder to stay neutral, a reminder to turn off notifications, and a statement about what the study will be about. (Thank you to Hyowon Gweon's lab for providing these materials)
?. [Stimuli Set Up]
Consent Slide: This slide is used to obtain consent from the parents. This can be updated depending on your IRB. We use, "I have read and understand the consent document, I am this child's parent or legal guardian and we both agree to participate in the study. It helps to have the part you want them to read outloud in quotation marks.
?. [Stimuli Set Up]
Soundcheck slide: any short, child-friendly video can be used here. See attached keynote and PowerPoint for one that you can use.  This is used to make sure volume is at a good level and videos are working properly. It also helps to have this video when babies are getting antsy. You can either have parents minimize their video here or make a separate video set-up slide (see 2.4).
?. [Stimuli Set Up]
Video Set up Slide: You can also add slides to help the parents set up their machines. There are several ways to set-up a synchronous study. The section below will explains one way.   [needs to be updated with graphics etc]
TITLE: Chlamydia trachomatis PCR
AUTHORS: Ana Ximena Kiguen, Jessica Paola Mosmann, Raul Fernando Venezuela, Cecilia Gabriela Cuffini

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify"><span>OmpA gene PCR: PCR DNA extract (5 μl) was used to amplify a 1087 pb fragment of the ompA gene of </span><span style = "font-style:italic;">C. trachomatis</span><span>, using primers NRO (5’CTCAACTGTAACTGCGTATTT3’) and NLO (5’ATGAAAAAACTCTTGAAATCG3´). PCR amplification processes commenced with a 4-minute denaturation step at 95°C and continued with 49 amplification cycles. Each cycle consisted of a first denaturation step at 95°C for 1 min, an annealing step at 55°C for 1 min and a final step of chain elongation at 72° C for 1.5 min. </span></div><style>
							  .justify:after {
							    content: "";
							    display:inline-block;
							    width: 100%;
							  }
							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify"><span>Cryptic Plasmid PCR: The primers used to generate a 201-bp fragment from the cryptic plasmid of </span><span style = "font-style:italic;">C. trachomatis</span><span> were CTP1 (5'-TAGTAACTGCCAClTCATCA-3') and CTP2 (5'-TTCCCCTTGTAATTCGTTGC-3'). The PCR amplification consisted of DNA denaturation at 95°C for 4 min followed by 35 cycles of amplification. Each cycle consisted of 1 min at 95°C, 1 min at 55°C and 1.5 min at 72°C followed by a final elongation at 72°C for 4 min. The ompA gene and cryptic plasmid PCR products were visualized after electrophoresis in a 1% agarose gel by ECO-Gel 20.000X Highway staining. Positive and negative controls were used in all determinations of PCR.</span></div><style>
							  .justify:after {
							    content: "";
							    display:inline-block;
							    width: 100%;
							  }
							</style></div></div>

[STEPS]
?. Pre Amplification Mix( NRO/NLO for ompA gene, and CTP1/CTP2 for cryptic Plasmid)
[Mint Master Mix]
[Agua ultrapura]
[each primer]
?. Add Extracted DNA
[DNA]
?. In a thermocycler , incubate as follows for ompA gene:49 amplification cycles as follows:Incubate as follows for Cryptic Plasmid:35 amplification cycles as follows:followed by a final elongation
?. Visualization of PCR products in 1.5% agarose gel
[Agarose]
[Water]
[Eco Gel]
TITLE: Viral Preparation
AUTHORS: Aditya Mohan

[STEPS]
?. [Day 1]
Coat the plates with 1% Poly-L-Lysine solution and leave in dry incubator overnight.
?. [Day 2 ]
Plate 6E6 HEK293 T cells overnight in DMEM media
?. [Day 3]
In 2 1.5 mL epindorph tube add 150 uL of OptiMEM. In one of them add 15 uL of lipofectamine. In the other add 10 uL of packaging plasmid and 10 uL of the plasmid of interest.
?. [Day 3]
Incubate for 5 minute.
?. [Day 3]
Combine the two tubes together for total volume ~ 300 uL and incubate for 5 minutes
?. [Day 3]
Remove the media from the plate and add the 300 uL dropwise. Add 1 ML of plain DMEM.
?. [Day 3]
Leave in incubator for 30 minutes
?. [Day 3]
Add 5 mL of DMEM based media.
?. [Day 5]
Remove the media and add to an eppendorf tube stored in 4 degrees. Add another 5 mL of DMEM based media.
4 °C
?. [Day 7]
Collect all the media.
TITLE: Size selection (Purification)
AUTHORS: Tsu-Chun Hung

[DESCRIPTION]
Size selection (Purification)

[STEPS]
1. Prepare 20 µL sample and 9 µL magnetic beads in 1.5 mL eppendorf tube.

 

2. Mix sample and beads gently by flicking then flash spin the tube. Put on the regular rack for 5mins 5 min.
 

3. Transfer the tube to the magnetic rack. After most of the magnetic beads attach to the wall, remove the supernant.
4. Add 300 µL 75% ethanol, flip whole magnetic rack around. Wait for 3 mins 3 min and remove the supernant.

5. Repeat step 4.
6. Quick spin the tube, and put on the magnetic rack. Remove superfluous solution with 10 μl pipette.

 

7. Add 10 µL elution buffer. Mix gently by flicking and flash spin the tube. Put on the regular rack for 10mins 10 min.

 

8. Transfer the tube to the magnetic rack. After most of the magnetic beads attach to the wall, collect the supernant to 200 μl PCR tube or 8-strip PCR tube.
9. Ready for 2' PCR.
TITLE: Reverse transcription, primer pools preparation and multiplex PCR steps for DENV1 serotype for genomic sequencing
AUTHORS: Laís Ceschini, Carla Julia da Silva Pessoa Vieira, Luisa Maria Inácio da Silva, Gustavo Lima, Raul Emídio, Tiago Graf, Gabriel Luz Wallau

[DESCRIPTION]
This step-by-step protocol describes the cDNA synthesis, primer pools preparation and multiplex PCR conditions with the main goal to sequence the complete genome of DENV1 serotype strains.

[STEPS]
SECTION: Reverse transcription
1.  Using a 2mL tube prepare the Mix 1 described below for 96 samples:



 



SECTION: Reverse transcription
2. Using 0,2mL PCR tubes or 96 wells plates add 11-16µL of extracted RNA from
RT-PCR positive samples. Add 2µL of Mix 1 to the tube/well and take it to the
thermocycler with the following set up

65ºC ---- 5 minutes
SECTION: Reverse transcription
3. Take the tubes/wells to ice for 1 minute (you can prepare a water bath with ice cubes to have a uniform temperature distribution).

SECTION: Reverse transcription
4. Using a 2mL tube prepare Mix 2:


 

SECTION: Reverse transcription
5. Add 7µL of Mix 2 to the tubes containing the Mix 1 plus RNA and take it to the thermocycler following the set up below:

Step1:
42ºC ---- 50 minutes
70ºC ---- 10 minutes
4ºC ---- Hold
SECTION: Reverse transcription
6. Store the cDNA at -20ºC.

Observation:. As a suggestion, to improve the final results only samples RT-PCR positive showing a Ct value of < 30 should be used for cDNA conversion and genomic amplification.

SECTION: Pools of primers
7. Select two 0,6mL tubes for each pool.
SECTION: Multiplex PCR
11. Prepare the Mix 1 for a Multiplex PCR for each Pool 1 and Pool 2 using a Falcon tube
of 15mL (~96 amostras) or a 2mL tube.


 

SECTION: Pools of primers
8. Using the original 100uM primer solution eluted individually, put them together following the table below containing each primer volume.
SECTION: Pools of primers
9. Pool 1 will have a final volume of 130µl and pool 2 of 170µl.
SECTION: Pools of primers
10. In order to prepare the solution to use in the Multiplex PCR, dilute each pool 1:10. That is, 10µl of pool 1 and 90µl of ultrapure water.

TABLE 1: Primers and pool order


 
*approximate concentration of each primer in the 25µl PCR reaction.

Note: The primers were designed using the https://primalscheme.com (Brito, 2021) based on
the JX669463.1 and KP188568 reference genomes.
SECTION: Multiplex PCR
12. Add 2,5µl of cDNA (totalling 5µl) in 22,5µl of the pool1 and pool2 reaction and take
it to the thermocycler following the conditions bellow:

Step1:
98ºC ---- 30 seconds
Step2: (45 cycles)
98ºC ---- 15 seconds
58ºC ---- 30 seconds
72ºC ---- 5 minutes
Step3:
72ºC ---- 2 minutes
Hold 4ºC
TITLE: Isothermal Titration Calorimetry of the Rubicon RH domain and Rab7
AUTHORS: Dan Tudorica

[DESCRIPTION]
1:1 binding of Rubicon and Rab7

[STEPS]
SECTION: Protein prep
1. On day prior to experiment, codialyze both binding partners to use in the the same beaker.
SECTION: Protein prep
2. Prepare 2 L of 50 mM HEPES 7.5, 150 mM NaCl, 10 mM TCEP, and degas thoroughly at room temperature under vacuum with stir bar agitation, then chill in fridge until ready for dialysis.
SECTION: Protein prep
3. Calculate sufficient protein for the assay: for a single run, want ~ 100 uL of 1 mM Rab7, and ~500 uL of 100 uM MBP-RUBCN RH domain. Samples can be more dilute, but should have enough total mass of protein to concentrate down later.
SECTION: Protein prep
4. In seperate dialysis bags, dispense purified Rab7 and RUBCN RH. Clip the ends of the dialysis bags twice, and place both dialysis bags in a 2 L beaker of the degassed and chilled dialysis buffer.
SECTION: Protein prep
5. Incubate for 24 H
6. After dialysis has completed, recover the protein samples and concentrate down to 1 mM for Rab7 and 100 uM for Rubicon. Spin protein samples at max speed on a tabletop centrifuge in order to pellet any protein aggregates, and allow protein samples to come to room temperature on a benchtop. Samples are now ready for ITC.

Note, changes in sample temperature can encourage the evolution of bubbles, which severely damages the quality of your ITC run. Make sure centrifuges are pre-warmed to room temperature, and keep all samples on benchtop rather than in ice bucket until ITC runs are completed.
SECTION: Isothermal Titration Calorimetry
7. In a Malvern PEAQ-ITC, set the resting temperature to room temperature, here ~ 22 C
SECTION: Isothermal Titration Calorimetry
8. Prepare for run by loading the chamber with ~ 300 uL of the Rubicon RH stock, and follow the automated syringe loading protocol to load the syringe with ~75 uL of the Rab7 stock. Gently 
SECTION: Isothermal Titration Calorimetry
9. Used the following parameters for this run, though changes can be made to suit your particular experiment

Reference power :41.9 uW
Temperature: 25 C
Stir speed: 750 rpm
Initial delay: 60 sec
Injection parameters: 13 injections, 3 uL each, 6 second duration, spaced by 150 sec. Initial inject was 0.4 uL with a duration of 00.8 sec, and not included in the analysis

Initiate run. Make sure that power baseline nears the reference power during the equilibration phase. If it does not, it is highly likely that a bubble was introduced, and the run should be aborted and the chamber reset. 
SECTION: Isothermal Titration Calorimetry
10. Open the native Malvern analysis software and visually inspect the automatically fitted baselines for quality.
SECTION: Isothermal Titration Calorimetry
11. Generate curve of best fit using the software and export. Inspect c-value of curve, and ensure that it falls within 10-100 in order to get an accurate KD determination.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>A protocol used to generate a PDMS microfluidic chip. Work was funded by Cambridge Synthetic Biology Strategic Research Initative (SRI) SynBio Fund.</p>
<p>http://www.synbio.cam.ac.uk/synbiofund</p>
<p> </p>

[BEFORE_START]
<ol>
<li><span style="line-height: 16px;">Use AutoDesk AutoCAD to design the microfluidic pattern printed on a photolithography mask.</span></li>
<li>
<p lang="en-GB">Order photolithograph masks from Microlitho Services (<a href="http://www.microlitho.co.uk/" target="_blank">http://www.microlitho.co.uk/</a>)</p>
</li>
</ol>
<p> </p>

[GUIDELINES]
<p> </p>
<p class="western" lang="en-GB">Materials</p>
<ul>
<li>
<p lang="en-GB">SU-8 photoresist (MicroChem, SU-8 2025)</p>
</li>
<li>
<p lang="en-GB">Propylene glycol monomethyl ether acetate (PGMEA; Sigma-Aldrich, cat. no. 537543)</p>
</li>
<li>
<p lang="en-GB">Poly(dimethyl siloxane) (PDMS) and curing agent (Dow Corning, Sylgard 184)</p>
</li>
<li>
<p lang="en-GB">Silicon wafers (3-inch diameter, Type-P, 1S polished; University Wafer, cat. no. S3P01SP)</p>
</li>
<li>
<p lang="en-GB">Isopropanol (Sigma-Aldrich, cat. no. 278475)</p>
</li>
<li>
<p lang="en-GB">1H,1H,2H,2H-Perfluorododecyltrichlorosilane (Sigma-Aldrich, cat. no. 729965)</p>
</li>
<li>
<p lang="en-GB">HFE-7500 (3M, cat. no. 98-0212-2928-5)</p>
</li>
<li>
<p lang="en-GB">Pico-Surf (TM) 1, 10ml, 5% in Novec 7500 (Dolomite, cat. No. 3200214)</p>
</li>
<li>
<p lang="en-GB">Miltex® BIOPSY PUNCH WITH PLUNGER 1.0mm (Williams Medical Product Code D6345 Mfg. Code SCH-33-31AA-P)</p>
</li>
<li>
<p lang="en-GB">Hamilton gas-tight syringe, 0.5 ml (Hamilton, cat. no. 201300)</p>
</li>
<li>
<p lang="en-GB">TUBING,POLY,20PE,30m (Harvard apparatus, cat. no.<span lang="en-US"> 59-8324</span>)</p>
</li>
<li>
<p lang="en-GB">Petri dishes (100 mm diameter × 15 mm</p>
</li>
<li>
<p lang="en-GB">Glass slides (75 × 50 mm)</p>
</li>
<li>
<p lang="en-GB">Scalpel</p>
</li>
<li>
<p lang="en-GB">Sharp tweezers</p>
</li>
</ul>

[STEPS]
?. 
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?. 
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?. 
?. 
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?.
TITLE: Biochemical Measures of Neuropathy - Western Blot Stripping
AUTHORS: Eva Feldman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">Oxidative stress is highly correlated with the metabolic changes caused by hyperglycemia. Increased levels of glucose overload mitochondria and result in the production of reactive oxygen species (ROS). In addition, the flow of excess glucose through cellular pathways decreases the cell’s normal ability to detoxify ROS. As a result, the neurons and axons of the peripheral nervous system contain increased levels of ROS and decreased antioxidant capacity. The following assays are used to measure these changes in rodent models of diabetic neuropathy. </div><div class = "text-block"><span style = "font-weight:bold;">Diabetic Complication:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/biz84kcn.jpg" /></div></div>

[STEPS]
?. [Performing assay:]
Rainbow markers do not withstand stripping, so if you don’t have a biotinylated marker on your blot, be sure to mark the location of the rainbow markers with a pen or pencil before stripping 1. Incubate your blot for 10-20 min at 70ºC. to strip. (10 min. for 20-40 µg protein and 15-20 min. for loaded protein over 40 µg or a very strong antibody) 2. Quick rinse in TBST. 3. Rinse 3 X’s 10 minutes each in TBST. 4. Re-block blot. 5. Run a loading control after each stripping.
?. [OR]
1. Incubate membrane in 0.5% Triton X-100 solution for 30 min. at room temp on a rocker. 2. Rinse 3 X’s 10 minutes each in TBST. 3. Re-block blot.
TITLE: Eggs injections
AUTHORS: FishFloorUCL

[DESCRIPTION]
Protocol for microinjections of 0-dpf eggs @FishFloorUCL.

[STEPS]
SECTION: Set-up breeding pairs
1. Set up the breeding pairs the night before or order them from the fish facility. Arrive early in the morning, 9AM for single cell, 9:30 for fish facility injections.

Make sure to book the injector in advance.
SECTION: Calibrate the needle
2. Open the red valve for your injector, you should see the dial turn to ~40
SECTION: Calibrate the needle
3. Power on switch
SECTION: Calibrate the needle
4. Set the time to 14-15ms
SECTION: Calibrate the needle
5. Take a needle from the large Petri dish on the side and stick in the blue tack above the microscope
SECTION: Calibrate the needle
6. Add what you want to inject (less is better, eg: 1-3 nL)
SECTION: Calibrate the needle
7. Place the black measuring slide under the microscope, add a drop or two of mineral oil and focus on the little ruler.
SECTION: Calibrate the needle
8. When tilting the whole needle base use both hands. When not using make sure it is tilted up
SECTION: Calibrate the needle
9. Tilt the needle into the oil
SECTION: Calibrate the needle
10. Break the needle with forceps and press the pedal until the correct amount is coming out of your needle (using the table on the wall to calibrate)
SECTION: Collect eggs
11. Take the dividers out of the fish boxes
SECTION: Collect eggs
12. wait 6-7 minutes
SECTION: Collect eggs
13. Transfer the fish to new boxes and replace the dividers for another round of eggs
SECTION: Collect eggs
14. Collect the eggs by pouring the water through a small sieve and transferring this to a Petri dish, clearly labelled 
SECTION: Inject the eggs
15. Use a Pasteur pipette to transfer eggs to a new pipette 
SECTION: Inject the eggs
16. Use a thin ended pipette to remove all the water and then line them up along the edge of a glass slide placed in the Petri dish
SECTION: Inject the eggs
17. Go down the line of cells and inject one at a time
SECTION: Inject the eggs
18. Collect a new clutch and repeat.
SECTION: Turn everything off/clean after yourself
19. Turn off the red tap.
SECTION: Turn everything off/clean after yourself
20. Turn time to minutes, press pedal until the dial has run completely down, flick it back to ms
SECTION: Turn everything off/clean after yourself
21. Turn off the power switch 
SECTION: Turn everything off/clean after yourself
22. Wipe down everything with ethanol.
TITLE: MycoEd Lab: CU Denver Protocols
AUTHORS: Andrew W. Wilson, sara.branco

[DESCRIPTION]
These lab protocols were written for the Fall 2023 Mycology course at University of Colorado Denver, Department of Integrative Biology instructed by Sara Branco and Andrew Wilson. The course is designed to guide students through an in-class semester long project that isolates fungi from plants, assess phenotypic variation using culture assays, extract DNA for genotyping/identification, harvest mycelium for genomic sequencing. Additional exercises in bioinformatics use phylogenetic analysis and online tools for gene assessments in fungi. The course ends with the students producing a scientific research poster based on their results.

[STEPS]