eamag/protocols-with-steps huggingface dataset:
TITLE: Citrate-Phosphate Buffer
AUTHORS: Neilier Junior

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A buffer solution has the function of resisting changes in pH even when adding powerful acids or bases. However, in the physiological environment the buffered system also provides cofactors for enzymatic reactions, critical salts and even essential nutrients for cells and tissues. Therefore, when trying to reproduce biological conditions in vitro, we must make the appropriate choice of the buffer. After all, it will provide the appropriate medium in which reactions will occur.</div></div>

[STEPS]
?. [Citrate-Phosphate Buffer]
Mix citric acid and sodium phosphate solutions in the proportions indicated and adjust the final volume to  with deionized water.  ABCDEFGHIJKLM1mL of Citric acid44.639.835.932.329.426.724.322.219.716.913.66.52mL of Sodium phosphate5.410.214.117.720.623.325.727.830.333.136.443.63pH2.63.03.43.84.24.65.05.45.86.26.67.0
pH range:   to  
(a) 0.1 M Citric acid; 19.21 g L-1 (M.W. 192.1 g mol-1)(b) 0.2 M Dibasic sodium phosphate; 35.6 g L-1 (dihydrate; M.W. 178.0 g mol-1) or 53.6 g L-1 (heptahydrate; M.W. 268.0 g mol-1)
100 ml
ABCDEFGHIJKLM1mL of Citric acid44.639.835.932.329.426.724.322.219.716.913.66.52mL of Sodium phosphate5.410.214.117.720.623.325.727.830.333.136.443.63pH2.63.03.43.84.24.65.05.45.86.26.67.0
?. [Citrate-Phosphate Buffer]
Adjust the final pH using a sensitive pH meter
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><span lang="EN-US" style="margin: 0px; line-height: 200%;">Increasing energy expenditure by stimulating thermogenesis through activation of brown adipose tissue (BAT) and/or induction of browning of white adipose tissue (WAT) is considered a promising strategy to treat/prevent obesity and related metabolic diseases</span><span lang="EN-US" style="margin: 0px; line-height: 200%;">. Whereas WAT is adapted to store energy as triglycerides, BAT produces heat (non-shivering thermogenesis). In brown adipocytes, the uncoupling protein-1 (<em>Ucp-1</em>) regulates conversion of energy into heat by uncoupling ATP production from mitochondrial respiration</span><span lang="EN-US" style="margin: 0px; line-height: 200%;">. Also in WAT adaptive <em>Ucp-1</em> positive adipocytes (brown in white: brite or beige) can arise, predominantly in subcutaneous (s) WAT</span><span lang="EN-US" style="margin: 0px; line-height: 200%;">. Therefore, this browning of sWAT can be visualized by making sWAT protein extracts and by analyzing protein levels of brown adipocyte-specific markers such as <em>Ucp-1</em> and peroxisome proliferator-activated receptor gamma coactivator 1 α (<em>Pgc1α</em>) in these extracts. </span></p>

[BEFORE_START]
<p><span lang="EN-US" style="margin: 0px; line-height: 107%;">A) Prepare Protein lysis buffer RL (10 ml):</span><br /><span lang="EN-US" style="margin: 0px; line-height: 107%;">-1 ml 100 mM Na<sub>2</sub>PO<sub>4</sub> (10 mM) /1.5 M sodium chloride NaCl (0.15 M)<br /> -400µl 25% Triton-X-100 (1%)<br /> -100µl 10% sodium dodecyl sulfate (0.1%)<br /> -1 ml 5% sodium deoxycholate (0.5%)<br /> -1 ml 2% sodium azide (0.2%)<br /> -6.5 ml MilliQ water</span></p>
<p> </p>
<p><span lang="EN-US" style="margin: 0px; line-height: 107%;">*Add fresh 1/100 EDTA and phosphatase and protease inhibitor cocktail (Halt phosphatase and protease inhibitor cocktail (100x), Thermo Fisher Scientific catalog number 78442) to buffer.<br /><br />B) Prepare NUPAGE LDS sample buffer supplemented with β-mercaptoethanol (20:1)<br /></span></p>

[STEPS]
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TITLE: Arabidopsis seeds priming
AUTHORS: Tao-Ho Chang

[DESCRIPTION]
The treated seeds can grow in medium or bulk soil to determine the impact of novel materials on plants. 

[BEFORE_START]
Seed priming is an important method that increases the health of the plant.

[GUIDELINES]
Seed treatment of arabidopsis seeds is a suitable, simple method for many plant-microbe interactions.

[STEPS]
SECTION: Seeds sterilisation
1. The arabidopsis seeds are immersed with 50% bleach in 1.5 µL tubes.
SECTION: Seeds sterilisation
2. 
  


SECTION: Seeds sterilisation
3. 100 rpm, 10 s, 28 °C 

SECTION: Seeds sterilisation
4. Gently remove the supernatant and leave the seeds in the tube.
SECTION: Seeds sterilisation
5. The sterile seeds are immersed in distilled water.
SECTION: Seeds sterilisation
6.   
Repeat the process of step #3 to #5 for 10 times

SECTION: Seeds treatment
7. The seeds were immersed in the specific concentration of treatment
1. Bacteria treatment: OD value to 0.1 and diluted 10 times.
2. Fungus treatment: final concentration of spore suspension is 100 spores mL-1.
SECTION: Seeds treatment
8.   

SECTION: Seeds treatment
9.   

SECTION: Wash seeds
10. The treated seeds are moved in a filter column with a collection tube.
SECTION: Wash seeds
11. 100 rpm, 10 s, 28 °C 

SECTION: Wash seeds
12. Remove the filter through in collection tube
SECTION: Wash seeds
13. Add 500 µL of distilled water to the filter column and resuspend the seeds.
SECTION: Wash seeds
14.   
Repeat the process of step #11 to #13 for 10 times
TITLE: Liver Tissue RNA Isolation - University of Minnesota TMCs
AUTHORS: Laura J Niedernhofer, David A Bernlohr

[DESCRIPTION]
InvitrogenTM TRIzolTM Plus RNA Purification Kit provides a simple, reliable, and rapid method for isolating high-quality total RNA from a wide variety of samples, including animal and plant cells and tissue, bacteria, and yeast.

This protocol was used to isolate RNA from liver tissue for BulkRNA sequencing in collaboration with The University of Minnesota Genomics Center. 

  

[STEPS]
SECTION: Isolate RNA
1. a. Lyse and homogenize samples in TRIzol Reagent by adding 1mL TRIzol reagent per 50-100mg tissue to the sample and use a homogenizer
b. Incubate for 5 minutes to permit complete dissociate of the nucleoproteins complex
c. Add 0.2mL of chloroform per 1mL TRIzol Reagent used for lysis, then securely cap the tube. 
d. Incubate for 2-3 minutes. 
e. Centrifuge the sample for 15 minutes at 12000 x g at4 °C - the mixture separates into a lower red phenol-chloroform, interphase and a colorless upper aqueous phase. 
f. Transfer ~600 μL of the colorless upper phase containing the RNA to a new tube
g. Add an equal volume of 70% ethanol, then mix well by vortexing. 
h. Invert the tube to disperse any visible precipitate that may form after adding ethanol. 


SECTION: Bind the RNA to the membrane
2. a. Transfer up to 700 μL of the sample to a spin cartridge with (collection tube).
b. Centrifuge 12000 x g for 15 seconds. 
c. Discard the flow through, then reinsert the spin cartridge into the same collection tube. 
d. Repeat step 2a -step 2c until the entire sample has been processed. 

SECTION: Wash the RNA on the membrane
3. a. Add 700 μL of Wash Buffer I to the spin cartridge. 
b. Centrifuge at 12000 x g for 15 seconds. 
c. Discard the flow-through, then reinsert the spin cartridge into the same collection tube. 
d. Add 500 μL of Wash Buffer II to the spin cartridge. 
e. Centrifuge at 12000 x g for 15 seconds. 
f. Discard the flow-through, then reinsert the spin cartridge into the same collection tube. 
g. Repeat step 3d-3f once. 
SECTION: Elute the RNA
4. a. Centrifuge at 12000 x g  for 1 minute to dry the membrane.
b. Discard the collection tube, then insert the spin cartridge into a recovery tube.
c. Add 30 μL-3 × 100 μL (3 sequential elutions with 100 μL each) of RNase-free water to the center of the spin cartridge.
- Note: If you are performing sequential elutions, collect all eluates in the same tube.
d. Incubate for 1 minute.
e. Centrifuge at >12,000 × g for 2 minutes.
f. Discard the spin cartridge.
The recovery tube contains the purified total RNA.
Store the purified RNA on ice if used within a few hours. For long-term storage, store the purified RNA
at –80°C.
If highly pure RNA without genomic DNA contamination is required, perform DNase I treatment after
purification (see PureLinkTM RNA Mini Kit User Guide (Pub. No. MAN0000406).
TITLE: Wet Chemistry RNA Isolation Method for Mature Cassava Leaf Tissue
AUTHORS: Lynn    Doran

[DESCRIPTION]
RNA extraction protocol for cassava for RNA of sufficient quality and quantity to perform RT-PCR gene expression studies.  

*Low recovery was achieved using Qiagen RNeasy kit. Typical recoveries using the mini protocol outlined here yielded 500-1300 ng/ul RNA from ~ 75 mg leaf tissue.*

[STEPS]
TITLE: Protocol 3: Growing liquid cultures of Agrobacterium prior to transformation day
AUTHORS: Sarah M Prostak, Edgar M Medina, Erik Kalinka, Lillian Fritz-Laylin

[DESCRIPTION]
Overnight liquid cultures of Agrobacterium transformed with the plasmid(s) of interest must be prepared the night before Spizellomyces transformation day. Liquid cultures must be inoculated from active plates/colonie. If you would like to use 25% glycerol stocks of previously transformed Agrobacterium, streak the desired cells onto the appropriate LB+selection plate, let it grow for 2-4 days at 28℃, then use this plate to inoculate the liquid media at least 12 hours before the intended transformation time. Ensure proper sterile technique throughout the protocol, using either a laminar flow hood or working in the sterile area around an open flame

[BEFORE_START]
Perform this protocol the night before your intended Spizellomyces transformation time.

[STEPS]
SECTION: Steps
1. Fill the appropriate amount of culture tubes each with 5 mL of LB liquid media.
 


SECTION: Steps
2. Using an inoculation loop or pipette tip, gently scrape the surface of the appropriate Agro plate to collect the bacteria or pick the colony of interest.
SECTION: Steps
3. Swirl the loop or tip to dislodge the bacteria into the media of the appropriate culture tube.
SECTION: Steps
4. Incubate at 28 °C, shaking at 225 rpm, 720 min, or  .
TITLE: Agarose gel pads for live Ashbya imaging
AUTHORS: ameya.jalihal  , Grace  

[DESCRIPTION]
A protocol to prepare agarose gel pads for live Ashbya imaging.

[BEFORE_START]
Rinse a depression slide with ethanol and have coverslips on hands.

[STEPS]
SECTION: Resuspension of Ashbya
1. 
 


SECTION: Resuspension of Ashbya
1.1. Spin down overnight Ashbya culture to pellet cells in 15 mL conical tubes 5 min 300 RPM



SECTION: Resuspension of Ashbya
1.2. Use a pipette to remove as much of the AFM as you can while leaving the cells at the bottom. Resuspend in 4 mL 1X LFM. Spin down again.  5 min 300 RPM. Either pipette pelleted cells from here onto the finished gel pad (steps 2.1-2.6), or do step 1.3 if the cells are too dense.
SECTION: Resuspension of Ashbya
1.3. Optional: Resuspend in 1 mL 1X LFM. Incubate on the rotator at 30 °C until ready to make gel pad, or for 30 min.
SECTION: Making agarose pads
2.   
 


SECTION: Making agarose pads
2.1. Aliquot  10 mL  of 1X LFM from step 1 into a 50 mL conical.
SECTION: Making agarose pads
2.2. Measure  200 mg  agarose. 
SECTION: Making agarose pads
2.3. Fill a 500 mL beaker with water to make a water bath and microwave the tube in 30 s increments until the agarose is dissolved. Shake the tube to dissolve agarose.

SECTION: Making agarose pads
2.4. Pipet 400 µL of the agarose solution into the depression on a depression slide. 
SECTION: Making agarose pads
2.5. Drag a coverslip across the depression to create the pad.  After about 2-5 min, gently lift the coverslip off. The gel pad is now ready to use. 




  
SECTION: Making agarose pads
2.6. When ready, add 50 µL of the cells to the center of the pad. Gently cover it with a coverslip, and press gently with a kimwipe to squish the cells.
SECTION: Making agarose pads
2.7. Stick a glass Pasteur pipette into a beaker of valap (you want to get the wax into the pipette). Then hold this over a Bunsen burner until the wax melts, and then apply around the edges of the coverslip to seal it:

  
The slide is now ready to image.
TITLE: Control light cycle and measure temperature/humidity in animal housing using Raspberry Pi and Python
AUTHORS: Matias Andina

[DESCRIPTION]
Python Code and Protocol for controlling animal housing lights, and measure temperature and humidity, using a Raspberry Pi, and ANAVI-light HAT.


[STEPS]
SECTION: Order parts
1. Order ANAVI LIGHT PHAT Advanced Kit (https://www.cro  wdsupply.com/anavi-technology/light-phat) and Raspberry Pi (compatible raspberry pi include RPi Zero W and RPi 3B+). This protocol has been tried using a Raspberry Pi 4.

SECTION: Order parts
2. Measure the length where you want to put your lights. 
SECTION: Install software
8. Go to house code repository at https://github.com/matiasandina/house    
SECTION: Install software
8.1. Read instructions and follow setup on the raspberry pi
SECTION: Order parts
2.1. Order LED strip according to length. It is recommended that you order an RGBW LED strip, as the white leds will be better for light than generating white light by driving the RGB to full power.
SECTION: Prepare and connect Hardware
5. Mount the ANAVI hat into the raspberry Pi
SECTION: Order parts
3. Order OLED Display Module (Amazon Link)  

SECTION: Order parts
4. Order a power supply that can provide at least 8V to the ANAVI LIGHT PHAT. 
SECTION: Prepare and connect Hardware
6. Feed the White wire from your RGBW into the BLUE socket of the ANAVI LIGHT PHAT. The code is expecting to output high through the blue pin. If you decide to go for the RGB version, you will need to modify the code.
SECTION: Order parts
4.1. Power supply can be fed through these DC adapter plugs  

SECTION: Prepare and connect Hardware
7. Connect temperature, light sensor, and OLED display to the I2C ports on the ANAVI LIGHT PHAT.
SECTION: Test
10. Test the functioning of the code (lights turn on, OLED display shows proper temperature and humidity). You can change default light hours and file issues to improve functioning. Inspect data folder to find the csv files with saved data
SECTION: Prepare and connect Hardware
9. Position lights and sensors in their desired place
TITLE: No Title
AUTHORS: 

[BEFORE_START]
<p>Prepare CTAB buffer and use within 1-2 weeks</p>
<p> </p>
<p style="margin: 0in; font-family: Calibri; font-size: 12.0pt;"><span style="font-weight: bold;">CTAB buffer (50 ml)</span></p>
<p style="margin-top: 7pt; margin-bottom: 0pt; font-family: Arial; font-size: 10.5pt; color: #333333;"><span style="background: white;">5ml Tris 1M, pH 8.0</span></p>
<p style="margin-top: 7pt; margin-bottom: 0pt; font-family: Arial; font-size: 10.5pt; color: #333333;"><span style="background: white;">2ml 0.5M EDTA</span></p>
<p style="margin-top: 7pt; margin-bottom: 0pt; font-family: Arial; font-size: 10.5pt; color: #333333;"><span style="background: white;">4.15g NaCl</span></p>
<p style="margin-top: 7pt; margin-bottom: 0pt; font-family: Arial; font-size: 10.5pt; color: #333333;"><span style="background: white;">1g CTAB</span></p>
<p style="margin-top: 7pt; margin-bottom: 0pt; font-family: Arial; font-size: 10.5pt; color: #333333;"><span style="background: white;">1g PVP</span></p>
<p style="margin-top: 7pt; margin-bottom: 0pt; font-family: Arial; font-size: 10.5pt; color: #333333;"><span style="background: white;">add ddH20 to 50ml, add stir bar, spin over low/med heat to dissolve, good on the benchtop for 1-2 weeks</span></p>

[GUIDELINES]
<p>Only attempted on Asparagus, Spirodela, and Milkweed. Works reasonably well. Uses a lot of ProtK and RNase H -- I have tested lower amounts with varying degrees of success. Some plants are just dirter than others, and overloading enzyme seems to help. </p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Ocular images play an essential role in ophthalmology. Current research mainly focus on computer-aided diagnosis using slit-lamp images, however few studies have been done to predict the progression of ophthalmic disease. Therefore by exploring an effective approach of prediction can help to plan treatment strategies and to provide early warning for the patients. In this study, we present an end-to-end temporal sequence network (TempSeq-Net) to automatically predict the progression of ophthalmic diseases based on consecutive slit-lamp images. First, we comprehensively compare six potential combinations of three convolutional neural networks and long short term memory (or recurrent neural network) in terms of effectiveness and efficiency, to obtain the optimal TempSeq-Net model. Second, we analyze the impacts of sequence lengths on model’s performance which help to evaluate their stability and validity and to determine the appropriate range of sequence lengths.</p>

[STEPS]
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TITLE: Visium CytAssist FFPE v4 -- University of Minnesota TMCs
AUTHORS: Laura J Niedernhofer, David A Bernlohr

[DESCRIPTION]
The Visium CytAssist Spatial Gene Expression for FFPE assay is designed to analyze mRNA in
tissue sections derived from formalin fixed & paraffin embedded (FFPE) tissue samples. The Visium
CytAssist instrument requires a glass slide with intact tissue sections as input. 

Tissue sections are processed with Visium Spatial Gene Expression assay performed by the University of Minnesota Imaging Center and sequenced by the University of Minnesota Genomics Center.



[STEPS]
SECTION: Deparaffinization, H&amp;E Staining, Imaging &amp; Decrosslinking
1.   

SECTION: Library Preparation &amp; Sequencing
2.   

 


SECTION: FASTQ Generation
3. BCL data from Illumina sequencer is demultiplexed and converted into FASTQ format using bcl2fastq software version 2.20.0
TITLE: Lean Start Keto Reviews &amp; Shark Tank 2022
AUTHORS: leanstartketoreviewsas  

[DESCRIPTION]
Lean Start Keto Reviews & Shark Tank 2022


[STEPS]
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What Are Spare Launch Keto Pills( spare launch keto capsules reviews) 
Spare Launch Keto Capsules are each-new 100 natural weight loss capsules that are manufactured by famed health experts in the United States with the end of helping the body burn fat for energy rather of carbohydrates. Spare Launch Keto Reviews by experts in the USA further described spare launch keto capsules as a weight loss formula that promises to help individualities in icing significant weight loss within a veritably short time. Spare Launch Keto Pills move the body into a state of ketosis to insure that its asked functions are carried out. Spare Launch Keto has been scientifically proven to be a dependable and effective way of dealing with unhappy weight gain. 

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TITLE: Designing Knockout Oligonucleotides (Instructor Protocol)
AUTHORS: Brian P Teague

[DESCRIPTION]
This is the instructor protocol for Designing Knockout Oligonucleotides, linked below.
 



[STEPS]
SECTION: Lab Setup
1. This is a "dry" lab - no pipetting involved. Each group of students will need a computer or a laptop.
SECTION: Lab Setup
4. If there are any genes that students should not choose, prepare this list in advance. In our case, students cannot pick any genes that have already been successfully knocked out. (Because many students don't move off of the first search results page, this includes most of those genes.)
SECTION: Instructor Tips and Common Student Errors
7. Instructor Tips
It helps to be familiar with Benchling -- there is a learning curve!
This works better if both students have a laptop and both are out -- one open to Benchling, and the other open to the instructions.
When Benchling has a split workspace, you can "drag" a tab from one side to the other.

SECTION: Instructor Tips and Common Student Errors
8. Common Student Errors
Not including the 1000 bp upstream and downstream of the gene in the import.
Forgetting to copy the reverse complement of the guide oligo (and instead copying the forward sequence.)
Not opening a split window for the CRISPR target selection. 
 

SECTION: Lab Setup
5. After the students have completed the lab, coallate all of the oligos (I use Benchling's Search function -- or, as above, ask students to copy into a shared folder.) If the section is large, it's difficult to completely double-check all of the oligos -- but I usually will check the following:
The "Guide" oligos are 26 bases long and start with "gacttt"
The "Guide RC" oligos are 26 bases long, start with "aaac" and end with "tt".
That the variable sequences of the guide and RC oligos are different.
The URA3 PCR F oligo is 59 bases long and ends with "tagtc"
The URA3 PCR R oligo is 57 bases long and ends with "attgg"
The KO PCR oligos are about 22 bp long and have a melting temperature of about 60.


SECTION: Lab Setup
2. Students will need accounts on Benchling before they dive in to this protocol. I use Benchling to run my entire lab section -- that's where protocols live and student notebooks are kept. I set up Benchling for each semester using this guide:

How to use Benchling for your teaching lab!

SECTION: Lab Setup
3. In particular, it is important that students share their individual projects with the instructor. This is so after they finish this lab, the instructor can find the oligos they created. (I also require my students to keep their notebooks on Benchling, so this facilitates my grading their notebooks as well. Alternately, you can ask them to copy the oligos into a shared folder.)
SECTION: Lab Setup
6. Don't forget to order the oligos! If you get them from IDT, they'll be ready to use within a week.
TITLE: Ethical Considerations and Compliance with Good Clinical Practice (Part 12 of 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 Part 12 of "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]