eamag/protocols-with-steps huggingface dataset:
TITLE: Colony PCR -- CHEM 584
AUTHORS: Ken Christensen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A quick way to screen transformants for an insert. This is faster and less expensive than doing a restriction digest.</div></div>

[STEPS]
?. [After you have colonies from a transformation]
Draw grid on clean agar plate (use the appropriate antibiotic). You will use this to inoculate an overnight culture for a positive clone.
?. [After you have colonies from a transformation]
Select colonies to pick. Streak portion of colony to numbered sector and place the remainder in a correspondingly numbered PCR tube with 50 µL of autoclaved water.
?. [After you have colonies from a transformation]
Heat at 95 °C for 10 minutes.** This can be done in PCR machine, a heat block, or a boiling water bath
?. [After you have colonies from a transformation]
Spin solution on high for 10 minutes to pellet cellular debris, and remove 4 µL for PCR.
?. [After you have colonies from a transformation]
Setup and run the PCR reaction, then assess the results using agarose gel electrophoresis. Be sure to include a positive control (e.g., plasmid) if available. Negative controls are also useful if one is available.
TITLE: Carbonate-Bicarbonate 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]
?. [Carbonate-Bicarbonate Buffer]
Mix sodium carbonate and sodium bicarbonate solutions in the proportions indicated.  ABCDEFGHI1mL of Sodium carbonate4.09.516.022.027.533.038.542.52mL of Sodium bicarbonate46.040.534.028.022.517.011.57.53pH9.29.49.69.810.010.210.410.6
pH range:   to  (a) 0.1 M Sodium carbonate (anhydrous), 10.6 g L-1 (M.W.: 106.0 g mol-1)(b) 0.1 M Sodium bicarbonate, 8.4 g L-1 (M.W.: 84.0 g mol-1)
ABCDEFGHI1mL of Sodium carbonate4.09.516.022.027.533.038.542.52mL of Sodium bicarbonate46.040.534.028.022.517.011.57.53pH9.29.49.69.810.010.210.410.6
?. [Carbonate-Bicarbonate Buffer]
Adjust the final volume to  with deionized water.
200 ml
?. [Carbonate-Bicarbonate Buffer]
Adjust the final pH using a sensitive pH meter.
TITLE: Bacterial Genomes Mining
AUTHORS: contact.microbialtec  

[DESCRIPTION]
The fast development of genome sequencing technologies and the power of advanced computational analyses of the DNA sequences dramatically increased the potential for natural product discovery. Our knowledge on the biosynthesis of secondary metabolites by bacteria is constantly expanding, thereby adding new information that connects biosynthetic genes with metabolites of particular chemical classes. With the cost-effective and rapid increase in the number of bacterial genomes sequenced, one challenge encountered in current drug discovery efforts is how to effectively prioritize those strains with the greatest ability to produce new compounds and understand the extent of biosynthetic potential that exists in nature.

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>Experiment purpose is to monitor the time-course of a large-scale infection of host cyanobacteria by phage under variable media conditions and obtain samples for proteomic and transcriptomic analysis.</strong></p>
<p> </p>
<p><strong>15 Hourly Timepoints: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 (unfiltered)</strong></p>
<p><strong>8 Even-hour Timepoints: 0, 2, 4, 6, 8, 10, 12, 14 (filtered)</strong></p>
<p> </p>
<p><em>Sampling is for monitoring the</em> <em>fluorescence vs. time and tracking the real-time progression of the phage infection.</em></p>
<p> </p>
<p><u>For Fluorescence measurement</u>, <strong>20 µl of sample</strong> was taken from each experiment bottle and diluted 10X in ASW in a 96-well plate for both filtered and unfiltered samples. The plate was read with the program “chla + phyco_onestep.prt” on the Tecan plate reader, which reads fluorescence at 680 nm (excitation 440 nm) for chlorophyll a and reads fluorescence at 577 nm (excitation at 544 nm) for phycoerythrin.</p>

[STEPS]
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?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This is the protocol for immunoprecipitation using Protein A/G Magnetic Beads.

[BEFORE_START]
Use 25 &micro;l of Protein A/G Magnetic Beads per 200&nbsp;&micro;l of crude cell lysate containing 200-500&nbsp;&micro;g of total protein in a standard immunoprecipitation protocol. It is important to increase the volume of beads proportionately for larger cell lysate volumes.

[STEPS]
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TITLE: Qubit (HS dsDNA Assay)
AUTHORS: Wolfram Moebius

[DESCRIPTION]
Qubit (HS dsDNA Assay)

[STEPS]
SECTION: Prepare working solution
1. 
NOTE: This is suitable for small numbers of samples (i.e. less than 24), including the quantification of the final libraries using the next generation sequencing SOP.

Label the required number of 0.5 ml tubes for Standard 1, Standard 2 and all your samples (use only the specific Qubit Assay tubes).
Label a 5 ml tube with ‘Qubit Working Solution’.

SECTION: Add samples to tubes and incubate
3. Alliquot190 µLQubit Working Solution into each of the tubes used for Standard 1 and Standard 2.
Aliquot198 µL Qubit Working Solution into each individual assay tubes
SECTION: Prepare working solution
2. Add 199 µL Qubit Buffer (either BR or HS, depending on the assay you want to do) to the 5 ml tube (n = samples + 2 standards + 1).
Add 1 µL Qubit Reagent (either BR or HS, depending on the assay you want to do) to the tube, mix by vortexing.
Tip: use a low vortex speed to avoid frothing; excessive frothing may require the preparation of additional diluted reagent to allow sufficient volume for all samples
SECTION: Add samples to tubes and incubate
4. Add 10 µLof Qubit Standard 1 and Standard 2 to the appropriate tube (the final volume will be 200 μl). Mix by vortexing 2-3 seconds, be careful not to create bubbles.
Add 2 µL stock DNA to the tube (the final volume will be 200 μl). Mix by vortexing 2-3 seconds, be careful not to create bubbles.
SECTION: Read standards
6. Select high-sensitivity double-stranded DNA assay on the Qubit and read the standards when prompted
SECTION: Read samples
7. Read samples when prompted (no need to re-read standards between readings) and record DNA concentration in ng/ul.
SECTION: Add samples to tubes and incubate
5.  
Incubate all tubes at room temperature for 5 min.
Tip: the fluorescence signal is stable for 3 hours at room temperature, so samples can be re-read if necessary
TITLE: 10x Genomics Library Construction
AUTHORS: Graham J Etherington, Darren Heavens, David Baker, Ashleigh Lister, Rose McNelly, Gonzalo Garcia, Bernardo Clavijo, Iain Macaulay, Wilfried Haerty, Federica Di Palma

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">10x Genomics Library Construction</div></div>

[STEPS]
?. Input DNA (in this case Polecat) was diluted down from  to  using Qiagen EB and checked using a QuBit 2.0 Fluorometer (Thermo-Fisher). The sample was then diluted in half as it was denatured. Finally,  of the diluted and denatured DNA was loaded onto the 10X chip which equated to .
2.5 µl
1.25 ng
?. Gel beads, oil and master mix were loaded into the appropriate wells on the 10X chip. The DNA was loaded along with the master mix. The chip was then loaded into the 10X Genomics Chromium instrument.
?. The formed emulsion was removed and placed into the thermocycler for  at . GEMs were cleaned up using the recovery reagent (10X Genomics), Silane beads (Thermo Fisher) and Tween-20 (Sigma-Aldrich) as per manufacturer’s instructions. DNA is cleaned up using a 0.7X ratio of SPRISelect beads (Beckman Coulter).
30 °C
?. The library underwent simultaneous End Repair and A tailing on the thermocycler at  for  and then  for . Adapters were then ligated at  for . A 0.8X ratio SPRISelect clean-up was performed. Sample Indexes were added during the final 8 cycle PCR amplification.  AB1  Temperature    Time  2  98°C (step 1)    45 secs  3  98°C (step 2)    20
  secs  4  54°C (step 3)    30 secs  5  72°C (step 4)    20
  secs  6  Steps 2-4  for 7 or 8 cycles in total       7  72°C (step 5)    60
  secs  8  4°C (step 6)    Hold
20 °C
65 °C
20 °C
AB1  Temperature    Time  2  98°C (step 1)    45 secs  3  98°C (step 2)    20
  secs  4  54°C (step 3)    30 secs  5  72°C (step 4)    20
  secs  6  Steps 2-4  for 7 or 8 cycles in total       7  72°C (step 5)    60
  secs  8  4°C (step 6)    Hold
?. A dual-SPRI size selection was performed at 0.5X then 0.7X ratios.
?. Library sizes were checked using the High Sense DNA bioanalyzer (Agilent). Molarity was checked using qPCR (KAPA Library Quant kit (Illumina), using ABI Prism qPCR Mix (Kapa Biosystems).
?. The library was clustered on the flow cell using the qPCR molarity. It was clustered at 8pM along with 1% PhiX spike-in (Illumina). Two lanes of a Rapid Run v2 flow cell were run on an Illumina HiSeq2500. 150bp PE, 8bp i7 index read, 0bp i5 index read. The libraries clustered at around 1100K/mm2. Manufacturer’s instructions: https://assets.contentful.com/an68im79xiti/4z5JA3C67KOyCE2ucacCM6/d05ce5fa3dc4282f3da5ae7296f2645b/CG00022_GenomeReagentKitUserGuide_RevC.pdf
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This procedure allows you to transform DNA into chemically competetent <em>E. coli</em>.

[BEFORE_START]
• Equilibrate a water bath to 42°C. <br />• Warm the vial of S.O.C medium to room temperature. <br />• Spread X-Gal onto LB agar plates with antibiotic, if desired for blue/white selection. <br />• Warm the selective plates in a 37°C incubator for 30 minutes (use one plate for each transformation). <br /><strong>IMPORTANT</strong>! It is essential that LB plates containing 100 μg/mL ampicillin are pre-warmed if you are performing the rapid chemical transformation procedure

[GUIDELINES]
Be extremely gentle when working with competent cells. Competent cells are highly sensitive to changes in temperature or mechanical lysis caused by pipetting. Transformation should be started immediately following the thawing of the cells on ice. Mix by swirling or tapping the tube gently, not by pipetting.

[STEPS]
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TITLE: Bird Measurements - ISL Peru
AUTHORS: Jorge Luis Mendoza-Silva, Cristian Tirapelle, Mrinalini Watsa, Gideon Erkenswick

[DESCRIPTION]
This protocol describes how to collect the relevant measurements during bird processing.

[GUIDELINES]
For an accurate description of measurements taken with birds, please refer to North American Banding Council Banders' Study Guide
https://nabanding.net/wp-content/uploads/2021/07/Banders-Study-Guide-2001.pdf

[STEPS]
SECTION: Lengths
1. If requested: measure the total length of the bird using a tape measure or callipers. Align the bird's neck and measure from the tip of the beak to the tip of the tail. Avoid stretching the bird's neck when you are doing this. 
SECTION: Lengths
2. The wing measurements are taken from the fold of the closed wing to the tip of the longest feather, first with a naturally positioned wing (wing chord)then with a flattened wing.

 
SECTION: Lengths
3. To measure the length of the tail, find the point of the rump and measure to the tip of the longest tail feather. 

  
  

SECTION: Bill measurements
4.1. The exposed culmen measures the distance between the tip of the bill and the edge of the feathering at the base of the upper bill.
  

SECTION: Bill measurements
5. head length is measured with a calliper from the tip of the beak till the back of the skull:

  
    
SECTION: Bill measurements
4.3. Bill width: measured with the calliper from side to side at the anterior end of the nostrils:

     

SECTION: Bill measurements
4. Bill measurements:
SECTION: Bill measurements
4.2. Culmen: from the opening of the nostrils to the tip o the bill

  

    

SECTION: Bill measurements
4.4. Bill depth: distance between the dorsal and ventral aspect of the beak, measured at the anterior end of the nostrils.
 
SECTION: Bill measurements
6. Tarsus length is measured with a caliper and it represents the distance between the depression in the intertarsal joint anteriorly, to the distal end of the last leg scale before the toes diverge distally.
TITLE: TissueCyte Troubleshooting Guide
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol outlines strategies to solve common problems that occur when setting up the TissueCyte 1000 system for normal operation.</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]
TITLE: Dynamic Light Scattering measurements
AUTHORS: arpine.sokratian

[DESCRIPTION]
Brief instrument guide for Dynamic Light Scattering Wyatt DynaPro followed by specific instructions to measure the size of sonicated amyloid fibrils

[STEPS]
SECTION: DLS overview
1.   

SECTION: DLS overview
2.   
  
  

SECTION: Sample preparation
3. Add 4 µLof  diluted sample (ranges  0.1-0.5 mg/mL ) onto   

SECTION: Sample preparation
4.   

SECTION: Sample preparation
5. Load the cuvette into the DLS machine 
  
  

SECTION: Recording the size distribution
6. Connect the device and the detector software
  

SECTION: Recording the size distribution
7. Record the 10 acquisitions for each measurement; 3 measurements for each sample
TITLE: Artificial Cerebrospinal Fluid VIII (ACSF.VIII)
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
This protocol describes how to make Artificial Cerebrospinal Fluid VIII (ACSF.VIII).  ACSF.VIII is used for multiple applications including incubation of fresh human brain slices prior to electrophysiological recording.

Note: 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.

[STEPS]
TITLE: Acute doses of beetroot juice supplementation on aerobic and anaerobic performance of trained male taekwondo athletes
AUTHORS: Hossein Miraftabi, Zahra Avazpoor, Erfan Berjisian, Amir Sarshin, Sajjad Rezaei, Raúl Domínguez, Reid Reale, Emerson Franchini, Mohammad Hossein Samanipour, Majid S. Koozehchian, Mark E.T. Willems, Ramin Rafiei, Alireza Naderi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Studies have shown that Nitrate (NO</span><span style = "vertical-align:sub;vertical-align:super;">3</span><span style = "vertical-align:sub;vertical-align:super;">-</span><span>) rich beetroot juice (BJ) supplementation improves endurance and high-intensity intermittent exercise. The dose-response effect on taekwondo of BJ supplementation are yet to be determined. This study aimed to investigate two acute doses of 400 mg of NO</span><span style = "vertical-align:sub;vertical-align:super;">3</span><span style = "vertical-align:sub;vertical-align:super;">-</span><span>(BJ-400) and (800 mg NO</span><span style = "vertical-align:sub;vertical-align:super;">3</span><span style = "vertical-align:sub;vertical-align:super;">-</span><span> (BJ-800) on taekwondo-specific performance tests compared with a placebo (PL) and control (CON) conditions. Eight trained male taekwondo athletes (age: 20±4 years, height: 180±2 cm, weight: 64.8±4.0 kg) completed four experimental trials using a randomized, double‐blind placebo‐controlled design: BJ-400, BJ-800, PL, and CON. Countermovement jump (CMJ) was performed before the Multiple Frequency Speed of Kick Test (FSKT) and Progressive Specific Taekwondo Test (PSTT), respectively, whereas cognitive function was assessed (via the Stroop test) before and after supplementation and 10 minutes following PSTT. Blood lactate was collected before the CMJ tests, immediately, and 3 minutes after the FSKT and PSST, with rating of perceived exertion (RPE) was recorded during and after specific taekwondo tests. Nosignificant difference between conditions were observed (p> 0.05)for PSTT and FSKT performances. In addition, blood lactate, RPE, heart rate, and CMJ height and performance during the specific taekwondo tests were not significantly different among conditions (p> 0.05). However, after the PSTT test, cognitive function was higher in BJ-400 compared to other treatments (p< 0.05). In conclusion, acute intake of 400 and 800 mg of NO</span><span style = "vertical-align:sub;vertical-align:super;">3</span><span style = "vertical-align:sub;vertical-align:super;">-</span><span>rich BJ did not improve taekwondo-specific performance tests in trained male taekwondo athletes.</span></div></div>

[STEPS]
?. 
?.
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<strong>Materials: </strong><br />• Sterile PBS <br />• Anti-human CD3 Antibody <br />       - Clone UCHT1 (LEAF™ format, Cat. No. 300413/300414/300432; Ultra-LEAF™ format, Cat. No. 300437/300438)<br />       - Clone OKT3 (LEAF™ format, Cat. No. 317303/317304/317315; Ultra-LEAF™ format, Cat. No. 317325/317326)<br />       - Clone HIT3a (LEAF™ format, Cat. No. 300313/300314; Ultra-LEAF™ format, Cat. No. 300331/300332) <br />• Cell culture medium (e.g., RPMI-1640 or IMDM supplemented with 10% FBS and 2mM L-glutamine) <br />• Sterile single-cell suspension of Ficoll-Hypaque-purified peripheral blood mononuclear cells, isolated T cells, or T cell subsets <br />• 96-well flat-bottom tissue culture plates with lids (e.g., Costar® Cat. No. 3596)<br /><br /><br />* Soluble forms of LEAF™ purified UCHT1 (1 µg/ml) or LEAF™ purified HIT3a (0.01 – 0.1 µg/ml) may be<br />used to activate T cells from PBMC cell populations.

[STEPS]
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TITLE: DAPI Staining Mouse Brain Sections
AUTHORS: daphne.toglia, Holly Myers

[DESCRIPTION]
This protocol details the steps for staining 4% PFA fixed mouse brain tissue sections with DAPI (4',6-diamidino-2-phenylindole), a fluorescent stain that can be used to label anatomic regions of interest in a mouse brain, allowing for imaging with a fluorescent microscope. The protocol includes a table with suggested staining duration based on section thickness and guidelines for calculating the volume of DAPI solution needed for staining. 

[BEFORE_START]
This protocol details DAPI staining for 4% PFA fixed mouse brain tissue that has already been sliced. Refer to protocol Sectioning Mouse Brain with Sliding Microtome for information about preparing slices ahead of DAPI staining. 

Throughout this protocol, tissue should be protected from light by covering well plate with aluminum foil between steps or equivalent protective measure.



[STEPS]
SECTION: Before Staining
1. Wash the mouse brain sections for at least 3 times, 5 min  each time in 1XPBS.


 

SECTION: Staining with DAPI
3. Pipette diluted DAPI (see note below) into each well with a manual pipette and place well plate on a shaker at Room temperature. Each well should contain enough DAPI solution to fully submerge the sections within. Leave well plate on shaker for suggested staining duration based on section thickness (see table below). Ensure that the well plate is covered in aluminum foil and away from direct light.  

 


 


 

SECTION: Staining with DAPI
4. After the incubation period, wash sections at least 5 times at 5 min each with 1xPBS. Refer to protocol Mounting and Coverslipping Mouse Brain Sections for next steps.
SECTION: Staining with DAPI
5. If unable to mount on the same day:
After 1X PBS washes, place sections in 1xPBS & 0.01% sodium azide solution, cover well plate with aluminum foil to protect from light, and store in 4 °C until ready for mounting.

SECTION: Diluting DAPI
2. 
Dilute 5mg/mL DAPI solution to 1:5000 with 1xPBS by measuring 1XPBS into a conical tube and adding DAPI with a manual pipette. Vortex DAPI before pipetting into 1XPBS.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes the affinity activity of microRNA93 through luciferase activity in 293T cells that have been transformed with a firefly luciferase coding sequence downstream of the BMP2 3'UTR. Using this methods we were able to idetificated microRNA93-5P binding the downstream 3'UTR of BMP-3 and inhibite the BMP-2 protein formation or not. </p>

[STEPS]
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TITLE: TS Spurrs - tissue fixed in Karnovsky&#39;s (TM - 013)
AUTHORS: sandra.crameri

[DESCRIPTION]
This method is used for conventional processing of tissue to Spurr's resin.

[GUIDELINES]
All time are minimum times, it is acceptable to go over time specified for any given step. Good place steps to leave overnight or at 70% ethanol and 50/50 Spurrs/Ethanol mix.

[STEPS]
SECTION: HEADER
1. SAN:


SPEC No:


OPERATOR & STEPS:


OPERATOR & STEPS:
SECTION: CONVENTIONAL
2. 2.5 % volume   plus 4 % volume    in 0.1 Molarity (m) for at least 40 min 

SECTION: CONVENTIONAL
3. Wash 0.1 Molarity (M) Sorenson's Phosphate Buffer pH 07.2 (300mosmol/kg) for 15 min 

SECTION: CONVENTIONAL
4. 1 % volume   in buffer for 60 min 

SECTION: CONVENTIONAL
5. 70 % volume   for at least 20 min 

SECTION: CONVENTIONAL
6. 95 % volume   for 20 min 

SECTION: CONVENTIONAL
7. 100 % volume   for 20 min 

SECTION: CONVENTIONAL
8. 100 % volume   dried molecular sieved for 20 min 

SECTION: CONVENTIONAL
9. 50 % volume   50 % volume   for 30 min 


SECTION: CONVENTIONAL
10. 100 % volume   30 min 

SECTION: CONVENTIONAL
11. 100 % volume   60 min 

SECTION: CONVENTIONAL
12. Oven polymerise overnight at 65 °C
TITLE:  Potassium phosphate buffer (1.0 M, pH 6.2)
AUTHORS: Andreas Sagen

[DESCRIPTION]
Potassium phosphate buffers are buffers with a buffering capacity between 5.8 and 8.0, where it is possible to adjust the pH and buffer strength with different ratios of Sodium phosphate monobasic and Sodium phosphate dibasic.

[STEPS]
SECTION: 500 mL Phosphate buffer (1.0 M, pH 6.2)
1. Fill a sterile 500 mL bottle with 400 mL distilled water
SECTION: 500 mL Phosphate buffer (1.0 M, pH 6.2)
2. Measure 12.035 g Sodium phosphate dibasic and 49.818 g Sodium phosphate monobasic

Materials:
 
 
SECTION: 500 mL Phosphate buffer (1.0 M, pH 6.2)
3. Add dry ingredients and mix for 5 min
SECTION: 500 mL Phosphate buffer (1.0 M, pH 6.2)
4. Fill to 500 mL with distilled water
SECTION: 500 mL Phosphate buffer (1.0 M, pH 6.2)
5. Filter sterilize through a 0.2 µm filter and store refrigerated (4 °C)
TITLE: Proteins annotation of Nano-DESI MSI datasets
AUTHORS: yang1832 , Julia Laskin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Scope: </div><div class = "text-block">Annotate protein species detected by Nano-DESI IMS analysis.</div></div>

[STEPS]
?. Create mass list from the averaged spectrum.
?. Calculate the charge state of each multiply charged peaks based on the spacing between the isotopic peaks.
?. Calculate the exact mass based on the m/z and charge state.
?. Obtain CID data from each sample.
?. Assign peaks by matching the exact mass and the CID spectrum to the protein databank.http://prospector.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msproduct