eamag/protocols-with-steps huggingface dataset:
TITLE: using a microwave instead of autoclave to sterilize bacterial culture media 
AUTHORS: Joseph Shenekji

[DESCRIPTION]
this protocol uses a microwave to sterilize and polymerize culture media for bacterial propagation in 3 minutes, the whole protocol can be done in 15-20 minutes without the need for an autoclave and waiting an hour for it to heat to 121 °Cwhich can consume a lot of electricity and time, this method is straight forward and clean and gave good results of growth without pollution of non desired bacteria, it can be used for liquid cultures and solid cultures as well.


[BEFORE_START]
prepare your media and bottle and plates, and make sure to turn on the laminar flow hood 15 minutes ahead of culture.

[GUIDELINES]
use heat gloves when you are taking the culture bottle out of the microwave 

[STEPS]
SECTION: adding the media powder
1. measure the desired quantity of media powder you want with a sensitive scale, ex: 4 g of  inside a heat resistant glass bottle with a lid with proper volume (size 250ml).
 


SECTION: adding the media powder
2. add the water you need, preferably distilled but tap water works fine for me, ex:100 mL.

SECTION: adding the media powder
3. close the lid tightly to create pressure, and mix by shaking in your hand for 30 s until the powder is dissolved.

SECTION: adding the media powder
4. put the media in the microwave n the highest degree for 3 min, stop the microwave if you see bubbles appearing, let the bottle rest for few seconds then continue the process.

SECTION: adding the media powder
5. you may need an extra minute to make sure everything is correct, the media will look yellow and transparent.
SECTION: adding the media powder
6. take out the bottle with the media and let it cool under the flow of tap water, be sure to rotate the bottle under water until it becomes easy to touch with your hand, that means it reached 60-65 °C.

SECTION: adding the media powder
7. sterilize the bottle by rubbing alcohol on it, and take it inside the laminar flow hood.
SECTION: adding the media powder
8. add your desired antibiotic like ampicillin  (10 µL )to the media and shake inside the hood gently to spread it.

SECTION: adding the media powder
9. pour the media inside petri dishes, about 10-15 mL is enough, then wait for about 15 min until it cools off and solidify.

SECTION: adding the media powder
10. culture on your plates containing the sterilized media with the microwave method, then close the plates and place them in an incubator 37 °C to grow your culture for 16-24 hours.

SECTION: results are here shown after 24 hours of growth
11. you can visually see the solid LB agar plates with grown cultures and no pollution of other bacteria.

 


SECTION: results are here shown after 24 hours of growth
12. i also tried a liquid LB broth and it gave good results with no pollution with very good growth.
TITLE: Hippocampal Neuronal Culture
AUTHORS: Daehun Park, Pietro De Camilli

[DESCRIPTION]
This protocol describes the procedure for hippocampal neuronal cultures from new - born mouse pups.

[STEPS]
SECTION: Protocol
1. Coat MatTek dishes with 1.5 mL of PDL solution for 180 min at 37 °C.

SECTION: Protocol
2. Wash dishes twice with culture grade water and incubate the dishes at 37 °C for 30 min.

SECTION: Protocol
3. Wash the dishes one more time and let dry. You can keep the dishes at 4 °C for few weeks (In this case, seal the dishes with paraffin film). 

SECTION: Protocol
4. Just before starting neuronal culture, prepare papain solution and leave at  37 °C water-bath.

SECTION: Protocol
5. Dissect hippocampi from P0 (postnatal day 0) new-born mouse brains using a stereo microscope. Collect tissue in ice cold complete HBSS.


 

SECTION: Protocol
6. Aspirate medium and wash 2-3 times with 10 mL fresh ice cold complete HBSS.

SECTION: Protocol
7. Add DNAse (final concentration: 20 μg/ml) to papain solution and filter sterilize. 
 


SECTION: Protocol
8. Add DNAse (final concentration: 20 μg/ml) to trituration solution and filter sterilize. 

SECTION: Protocol
9. Aspirate the enzyme solution and wash twice with trituration solution
SECTION: Protocol
10. Resuspend samples in 1 mL trituration solution. Gently dissociate neurons with  a P1000 filter tip by pipetting up and down for 10-12 times. Avoid generating any bubbles.

SECTION: Protocol
11. Count the cells.

 



SECTION: Protocol
12. Seed the cells on the PDL coated MatTek dishes in pre-warmed Plating Media (2 mL).

(0.4 million cells / one MatTek dish).

SECTION: Protocol
13. After 180 min incubation at 37 °C and 5% CO2, change the plating medium to  pre-warmed Complete Neurobasal Media.

SECTION: Protocol
14. Remove 500 µL of media and add 750 µL of fresh complete neuronal media at DIV (days in vitro) 4, 7 and 14. Incubate the media at 37 °C  before adding it to neurons.
TITLE: Pyrolysis kinetics
AUTHORS: Jiaxin Li

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The sensitivity of cells in planktonic cultures to phages was tested in 96-well microtiter plates.</div></div>

[STEPS]
?. A 1:100 dilution of overnight bacterial culture in LB should be prepared and incubated at 37°C to reach an optical density of 0.5 at 600 nm (OD600).
?. 100 µl aliquots of diluted culture were distributed in microplate wells and 100 µl single or mixed phage suspensions (at MOI 10; 1; 0.1 and 0.01) were added.
?. Microplates were incubated for a further 24 h and optical density was measured at 600 nm in a plate reader every 870s.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Phenol/chloroform-based DNA extraction from cells pre-treated with RNase A, lysozyme, proteinase K, and SDS. The protocol was optimized for extracting DNA from <em>Microcystis aeruginosa</em>, but works well for other cyanobacteria.</p>

[STEPS]
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TITLE: Paramecium cultures for RNAi 
AUTHORS: Ben Jenkins

[DESCRIPTION]
Filtering and preparing P. bursaria cultures for downstream RNAi

[STEPS]
SECTION: Choosing culture
1. Use an old culture of Paramecium bursaria (between one and six months old). 

# This is to ensure that cultures start from a vegetative growth stage
SECTION: Filtering
2. In a sterile flow hood, filter 100mL of P. bursaria culture through a 40um cell strainer (3x). Keep the flow-through.

# This removes large clumps and debris from the culture
# Remain in hood for the remainder of the protocol
SECTION: Filtering
3. Transfer P. bursaria to a fresh culture flask and leave for 1-2 hours.

# This allows cells to digest any residual food present in the food vacuole
SECTION: Filtering
4. Aliquot 5uL onto a slide and count cells (x10).

# This is to ensure the correct volume and cell number (~50 cells per well) is added
SECTION: Filtering
5. Pour P. bursaria into a sterile square petri dish.

# Place the lid of the petri dish under one side so that the petri dish is at an angle 
SECTION: Filtering
6. Using a multi-channel pipette, aliquot the calculated volume of P. bursaria into each well.

# The final cell number should be ~50 cell per well
# Shake the petri dish gently with your hand (two to three times), and then aliquot as quickly as possible
# Try not to pipette back and forth in the petri dish, as this can push the cells to the edge and create an uneven distribution of cells across the wells
SECTION: Filtering
7. Top up to 250uL per well with NCL.

# Autoclaved NCL with antibiotics added in the flow hood under sterile conditions:
# Ampicillin (0.1 mg/mL), IPTG (0.4 millimolar (mM)), and β-sitosterol (0.0008 mg/mL)
SECTION: Filtering
8. Add a lid to the plate and place at 23 °C 

# Or whatever condition required for the experiment
# Under standard conditions, ensure the plate is exposed to adequate light
TITLE: T-3 TICK STORAGE
AUTHORS: REDI-NET Consortium

[DESCRIPTION]
This protocol describes tick storage.

[GUIDELINES]
OBJECTIVE

To outline steps for properly storing field-collected tick samples and nucleic acid samples purified from these ticks.

SUMMARY/SCOPE

The overarching aim of the REDI-NET is to develop a collaborative laboratory network between domestic and international partnering institutions to address disease surveillance needs in order to effectively detect, predict and contain potentially emergent zoonosis. This SOP provides guidance on storage of tick samples and their purified nucleic acid to preserve their integrity for downstream nucleic acid extraction and/or sequencing library preparation.


MAINTENANCE OF EQUIPMENT

Decontaminate a PCR workstation by keeping the UV light on for 15 min.
 
 


[STEPS]
SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
1. 
 
Cool 96-well microfuge tube racks on ice.
SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
2. Using permanent markers, label 1.5 ml microfuge tubes with unique sample ID.
SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
3. Using clean forceps, transfer individual tick into the corresponding pre-labeled 1.5 mL microfuge tubes and put it onto the microfuge tube rack (on ice) sequentially.
SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
4. Once the rack is full or all tick samples have been completed, label the rack with a unique rack ID.
SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
5. Close the rack lid tightly, secure with clear Saran wrap and immediately transfer to -80 °C freezer.
SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
6. Update the freezer inventory so samples can be tracked properly.
SECTION: STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID
7. 
 
In the clean PCR workstation, carefully transfer the eluted total nucleic acid to a 96-well PCR microplate, make sure to keep samples in the exact same locations corresponding to the rack where the original ticks were stored.
 

SECTION: STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID
8. Cover the 96-well PCR microplate with adhesive film to prevent spill over or contamination.
SECTION: STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID
9. Label the film with a unique plate ID.
SECTION: STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID
10. Immediately transfer the 96-well PCR microplate to -80 °C freezer.
SECTION: STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID
11. Update the freezer inventory so samples can be tracked properly.
TITLE: Long staining procedure of nuclei in Euplotes crassus using DAPI
AUTHORS: Rachele Cesaroni

[DESCRIPTION]
<div class = "text-blocks"></div>

[STEPS]
?. Pellet Euplotes crassus cells at 400 rcf for 3 minutes, and remove as much supernatant as possible by pipetting.
Both algae and bacteria are autofluorescent. Better to have a completely starved Euplotes crassus culture.
?. Add 1 ml of 2% PFA in 1X PHEM or 4% PFA in 1X PBS to the cells, and incubate them for 10 minutes at room temperature.
?. Pellet Euplotes crassus cells by centrifugation at 400 rcf for 3 minutes, and remove as much supernatant as possible by pipetting.
?. Wash cells twice with 1X PBS (400 rcf for 3 minutes each time).
?. Add 1 ml of TBSTEM - 3% BSA and 0.5 μl of DAPI (0.1 mg/ml) to the cells, and stain for 10 minutes at room temperature.
?. Pellet Euplotes crassus cells by centrifugation at 400 rcf for 3 minutes.
?. Add 50 μl of Prolong medium.
?. Place an approx. 10 μl droplet of Euplotes crassus cells on a slide for observation by fluorescence microscopy.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong><u>HBV DNA Real Time Quantification according to ARNS 12187 project</u></strong></p>
<p> </p>
<p>Using the Arrow extractor (NorDiag, Biotrin International, Ireland), DNA was extracted from 240 μl of plasma, pre-treated with10 μl of proteinase K, using the Arrow Viral NA extraction kit, according to the manufacturer’s instructions during 45 min. Template DNA was eluted into 60 μl of kit elution buffer. Besides clinical samples, quantification standard (Acrometrix HBV Panel, Acrometrix, Menica, CA, USA) was also extracted for each run with the same protocol and1:10 diluted (from 50,000,000IU/mL to50IU/mL). For amplification, we used a primers/probe set designed under the auspices of “Agence Nationale de Recherches sur le SIDA et les hépatites virales” (ANRS 12187 project) and targeting a conserved region in the HBVS gene (nucleotide (nt) positions, 379–426). All runs were performed in a 50-μl volume containing DNA extract (10 μl), Master Mix (Platinum UGD, USA) (25 μl), pure water (HyClone Pure Water, Thermo Fisher Scientific, Waltham, MAUSA)(12.5 μl), forward primHBV1 (5’-GTGTCTGCGGCGTT TTATCA-3’) and reverse primHBV2 (5’-AGGCATAGCAGCAGGAT GAA-3’) primers at 10 μM (1μl each) and probe (5’FAM-TGCGGCGTTTTATCAT-MGB3’) at 5μM (0.5 μl). Each reaction consisted of: 2min at50°C and 10min at95°C; followed by 50 cycles of 15sec at 95°C and 1 min at 60°C each. The lower limit of quantification (LLOQ) of our technique was 100IU/mL and the lower limit of detection (LLOD) was 50IU/mL.</p>

[STEPS]
TITLE: Soil extract for algal media
AUTHORS: Roscoff Culture Collection

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">How to prepare soil extract</div></div>

[STEPS]
?. Sample 10 g of dry soil from a place that does not contain any pesticide nor pollutant and where you can be sure to be able to come back in order to always use the same soil. The soil must be dry because the boiling step will be longer if the soil is not dry.
?. Add the 10 g soil to 400ml of milliQ water.
?. Boil during 1 hour.
?. Filter on 0.2 µm or first through 0.8/0.4 µm and then through 0.2µm in order not to clog the filter.
?. Aliquot and freeze.
?. Add 30 à 50ml of soil extract per 1 L of medium.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Works requiring taxonomic knowledge face several challenges, such as arduous identification of many taxa and an insufficient number of taxonomists to identify a great deal of collected organisms. Machine learning tools, particularly convolutional neural networks (CNNs), are then welcome to automatically generate high-performance classifiers from available data.</p>
<p>We propose an ensemble of CNNs to identify ant genera directly from the head, profile and dorsal perspectives of ant images.</p>
<p>Transfer learning is also considered to improve the individual performance of the CNN classifiers.</p>

[STEPS]
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TITLE: KAPP-Sen TMC: Whole Pancreas Preparation
AUTHORS: Cristina Aguayo-Mazzucato, Kanako Iwasaki

[DESCRIPTION]
Whole pancreas and tissue blocks preparation

[STEPS]
SECTION: Overview
1. All the whole pancreases were procured by the University of Texas Health San Antonio and shipped to Joslin Diabetes Center, University of Harvard Medical. The pancreases were cut into the Head, Body, and Tail of the locations along the guideline (1), cut into 4mm thickness, and placed into order paraffin or frozen cassettes. The paraffin cassettes were fixed in 4% paraformaldehyde for 24 hours and processed for paraffin embedding at Joslin Histology Core. The frozen cassettes were molded with O.T.C. compound (Fisher Scientific No.23-730-571) and frozen in the cold 2-Methylbutane (Fisher Scientific No.60-048-072) bath followed by dried and stored at -80°C. At least three paraffin cassettes per each location of Head, Body, and Tail were shipped to The Jackson Laboratory; all the procedures were done at 4°C and avoided exposure to direct light.
SECTION: Preparation of Human Pancreas
2. 30 minutes before the pancreas arrives:
1. Prepare 2-Methylbutane bath
 -Fill the bottom of the plastic big beaker with dry ice (use tip, not powder dry ice)
 -Place a glass beaker inside a larger plastic beaker
 -Add dry ice around the glass beaker up to 400.
 -Pour 100% EtOH onto dry ice until it appears "slushy" - keep adding dry ice chips and ethanol as needed. Should be bubbling. 
 -Fill the glass beaker about half-way with 2-Methylbutane and let it chill for 15-20 minutes before using.
 
2. Prepare Cassettes
-Name the cassette "JDC-WP-###-X" to both green paraffin cassettes and clear frozen mold. #### should be the experimental number. X should be alphabetical from a to z. After z, create aa, ab, up to ae.
-Add OCT to cover the bottom of a labeled frozen mold without bubbles.
-Set everything like the pictures
 
3. When the Pancreas Arrives
-Get ice and place between two trays.
-Pour PBS into the top tray until the pancreas is completely immersed.
  4. Test isopentane to make sure that it is cold enough - with long forceps, put mold with OCT medium into chilled isopentane (if it doesn't turn within 5 seconds, wait longer, and add more dry ice and more ethanol).

5. After removing fat from the pancreas, spread out pancreas on wax, and cut into Head, Body, and Tail (see Section "Examination of Specimens from Patients with Carcinoma of the Pancreas" for the definition of anatomical locations), and measure the weight. 
    -Paraffin: put it in the green cassettes from 'a'
    -Frozen: put it onto the mold and add another OTC. Dry it by placing it on dry ice, and standing it up at an angle.
 
6. Be sure to bleach everything for over 30 minutes. Always treat it as contaminated with an infectious disease. 
  
      
SECTION: Examination of Specimens from Patients with Carcinoma of the Pancreas
3. From "Pancreas Exocrine 4.0.0.1" page 8, explanatory note B. Definition of Location
TITLE: Rotarod test  
AUTHORS: mariangela.massarocenere

[DESCRIPTION]
Protocol to assess motor coordination and balance by using an accelerating rod

[STEPS]
1. Habituate the animal for 1 h to the testing room before the test 
10. 
2. Place the rat on the elevated rod (47 cm from the floor) rotating at low speed (4 rpm) for at least 30 seconds before the first session of the test
3. Place the animal on an accelerating rod from 4 to 40 rpm in 300 s
4. Repeat step 3 for 3 times with at least 5 min of rest between trials
6. Repeat steps 4-5 for 4 times in two consecutive days (with an inter-session interval of 3.5 h)
4.1. Record the time the rat fall from the rod (maximum 300 seconds)
5. Clean the apparatus every time between animals and trials
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The Anti-Neu5Gc Antibody Kit contains the essential monospecific polyclonal chicken IgY antibody, along with a negative control primary antibody to detect the presence of Neu5Gc on glycoconjugates by Western blot (WB). Samples to be evaluated are first subjected to SDS-PAGE, followed by transfer to a nitrocellulose or polyvinylidenedifluoride (PVDF) membrane. The membrane is then incubated with affinity-purified polyclonal anti-Neu5Gc to determine the presence of Neu5Gc on the protein of interest.</p>
<p> </p>
<p>The antibody provided in this kit has been shown to identify as little as 5 pmol of Neu5Gc per ug glycoprotein, which is at or below the current detection limit for conventional analysis by acid release, purification, DMB derivatization, HPLC, and electrospray mass-spectrometry. The Western blot provides additional information in that it confirms that Neu5G is directly linked to the glycoprotein of interest rather than to an accompanying sample component.</p>

[GUIDELINES]
<p><strong>Items required but not supplied:</strong></p>
<p> </p>
<p>- Electrophoresis setup for SDS-PAGE and blotting</p>
<p>- Molecular weight maker</p>
<p>- Positive control protein</p>
<p>- Negative control</p>
<p>- TBS and TBS-T</p>
<p>- Enzyme-conjugated secondary anti-chicken IgY antibody (HRP or AP)</p>
<p> </p>

[STEPS]
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TITLE: (ILLUMINA MENU) Protocols for SARS-CoV-2 Library Prep with ARTIC Primers, Bioinformatic Analysis, and Database Submission 
AUTHORS: Technical  Outreach and Assistance for States Team

[DESCRIPTION]
This collection of protocols covers library preparation and sequencing on Illumina platforms as well as bioinformatic analysis and submission of SARS-CoV-2 data. These frequently used protocols have been assembled by the Technical Outreach and Assistance for States Team (TOAST) and the Office of Advanced Molecular Detection (OAMD) at the Centers for Disease Control and Prevention for use by participating State, Tribal, Local, and Territorial labs for sequencing of SARS-CoV-2. Individual protocols were developed by the protocol authors, and individual protocols should be credited accordingly. 

These documents are intended for laboratories designated by TOAST and should not be widely or openly disseminated.

  



For technical assistance, please contact: TOAST@cdc.gov

[STEPS]
TITLE: GST pull down assay
AUTHORS: minghao chen, Xuefeng Ren

[DESCRIPTION]
GST pull down assay

[STEPS]
1. Mix the GST tagged protein and fluorescent protein with 30 μl Glutathione Sepharose beads (Cytiva) at final 1 μM concentration in the buffer of 25 mM HEPES at pH 7.5, 150 mM NaCl, 1 mM MgCl2 and 1 mM TCEP.  The total volume is 200 μl. 
2. Rocking at 4 °C   

3. Wash the beads four times, then eluted in 50 μl of buffer with 25 mM glutathione 



4. Mix 18 μl eluent in lithium dodecylsulfate (LDS)/BME buffer and subjected to SDS/PAGE gel without heating samples. The gel was scanned at 488 or 550 nm in ChemiDoc MP imaging system (Bio-Rad).
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>This is a short tutorial on how to get started analyzing FASTA files via the command line.</strong></p>
<p> </p>
<p>Code is intended for use on an Ubuntu 16.04 LTS OS, but it may work on other Unix or Unix-like systems.</p>
<p> </p>
<p>Here we will mainly use standard Unix commands as well as the seqtk tool. </p>
<p>Information on seqtk, including information on installation, can be found here: https://github.com/lh3/seqtk</p>

[STEPS]
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TITLE: Concentrated DNA Loading dye stock: User protocol
AUTHORS: Stephane  Fadanka, Nadine    Mowoh

[DESCRIPTION]
Beneficial Bio 6X DNA Loading Dye is a premixed loading buffer used to prepare samples for
loading onto agarose and polyacrylamide gels. It contains bromophenol blue and xylene
cyanol dyes for visual tracking of DNA during electrophoresis. The trehalose ensures that
DNA forms a layer at the bottom of the well and facilitates sample loading. The EDTA
chelates metal ions and stops metal-dependent enzymatic reactions. The solution contains
SDS which results in sharper bands by removing restriction enzymes from DNA.

Features:

Double tracking: Xylene cyanol, Bromophenol blue
No interaction with bands during gel exposure to UV or Blue light
Inhibits metal-dependent nucleases (EDTA)

Applications:

Preparation of DNA ladders, markers, and samples for loading on agarose or polyacrylamide
DNA electrophoresis.

[BEFORE_START]
Ensure all materials, equipment and chemicals to be used for this experiment are all in place before starting. 

[GUIDELINES]
Follow good laboratory practices when carrying out experiment although all reagents used are generally safe.

[STEPS]
SECTION: User Protocol
1. 
 

Preparing 1ml of 6x working stock:

Vortex or shake the concentrated stock tube for 10 seconds prior to use.
Dilute 1 part of concentrated DNA Loading Dye stock with 9 parts  of 20% Trehalose (pipette 100 µL from the concentrated dye stock and add 900 µL of 20 % (v/v)Trehalose to make a 1ml 6x working stock).
Cork the tube firmly and mix by inverting the tube several times.
Store the working solution at room temperature or4 °C for up to 12months, for longer periods, store at-20 °C.
To use, dilute the nucleic acid solutions 1:5 to give a 1x solution e.g. add 1 µL 6x DNA loading dye for every5 µLDNA.
Load the sample into a precast agarose gel well, run to finish and visualize the gel with the help of a UV transilluminator or blue gel system.



SECTION: Notes and troubleshooting
2. BenBio 6X DNA Loading Dye enables easy tracking and sample migration and follow up during the run without creating background noise during visualization.
If you notice any interference of the tracking dye with the DNA bands, check the working concentration and try further dilutions to find the one that works for you.