eamag/protocols-with-steps huggingface dataset:
TITLE: Agrobacterium-mediated transformation of the chytrid fungus Spizellomyces punctatus (Sp)
AUTHORS: Sarah M Prostak, Edgar M Medina, Erik Kalinka, Lillian Fritz-Laylin

[DESCRIPTION]
This is a collection of protocols for Agrobacterium-mediated transformation of the chytrid fungus
Spizellomyces punctatus.

[GUIDELINES]
Please thoroughly read through each protocol entry before starting, including the materials, guidelines, and warnings

[STEPS]
TITLE: No Title
AUTHORS: 

[BEFORE_START]
<p>Be sure to mix and autoclave <strong>solution I</strong> and <strong>solution II</strong> separately before combining and adding trace metal solutions and vitamin solutions. Store media in 4ºC. Note, Vitamin stock solutions amounts are per mL Trace Metal stock solution amounts are per one liter. Adjust amounts, as needed, according to desired stock amount. </p>

[STEPS]
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TITLE: Universal sandwich ELISA for investigating the binding of avian and mammalian immunoglobulins  to Streptococcal protein-G (SpG) using a peroxidase-labeled Protein LAG conjugate (SpLAG-HRP).
AUTHORS: Angel  Justiz-Vaillant

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

[STEPS]
?. This ELISA is used to study the interaction of streptococcal protein-G (SpG) with different avian and mammalian immunoglobulins.
?. The 96 well microtitre plate is coated overnight at 4°C with 2 µg/µl per well of SpG in carbonate-bicarbonate buffer pH 9.6.
?. Then plate is treated with bovine serum albumin solution and washed 4X with PBS-Tween.
?. 50 µl of  avian egg yolk, egg white (1:8 dilutions) or 50 µl of sera or 50 µl of mammalian IgG (1mg/ml) is added to the well and incubated for 1.30h at room temperature and the microplate is then rewashed 4X with PBS-Tween.
?. Then 50 µl of peroxidase-labeled-Protein-LAG (SpLAG-HRP) conjugate diluted 1:15000 in PBS-non-fat milk is added to each well and  incubated for 1.30h at RT. The plate is washed 4X with PBS-Tween.
?. Pipette 50 μl of TMB  (Sigma-Aldrich) to each well.
?. The reaction is stopped with 50 µl of 3M H2SO4 solution.
?. The plate is visually assessed for the development of colour and read in a microplate reader at 450 nm.
?. A cut-off point should be calculated as the mean of the optical density of negative controls x 3. The higher the OD value the higher will be the affinity of SpG to immunoglobulins.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Congenital Zika Syndrome (CZS) is associated with microcephaly and other malformations of the central nervous system. We identified that some of areas damaged by CZS are also known to influence the neural circuitry controlling the lower urinary tract (1). And decided to investigate neurogenic bladder, in an attempt to identify risk factors and therapeutic modalities that could mitigate the impact of the CZS in infected children.</p>
<p>A research protocol was developed based on our experience treating neurogenic bladder in children. And, after ethical approval, tested during urological assessment in patients with confirmed microcephaly due to CZS who were referred to our pediatric urodynamics clinic between June 2016 and May 2017. The protocol consisted of obtaining clinical history, laboratory tests, renal and bladder ultrasound and a urodynamic evaluation. ZIKA virus was previously confirmed by maternal history and positive RT-PCR in babies/mothers. Microcephaly and other central nervous system abnormalities were established based on neurological and image evaluation (CT and/or MRI).</p>
<p>The results were submitted for publication and we are waiting the editor’s decision. Neurogenic bladder was confirmed so far in 100% of patients tested, with high-risk urodynamic patterns that may cause renal damage if left untreated. This is a preventable health condition in the setting of Congenital Zika Syndrome. While further investigation is necessary to understand long-term disease behavior, bladder proactive management is paramount to help mitigate disease burden for patients and their families.</p>

[GUIDELINES]
<p><strong> </strong></p>
<p><strong>Protocol Recommendation</strong>: all children with confirmed Congenital Zika Syndrome need to be refered for urological assessment</p>

[STEPS]
?.
TITLE: FLASH amp
AUTHORS: eesha.sharma.phd 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">FLASH amp is a method that allows for room temperature detection and amplification of a viral RNA sequence of interest submitted as part of the Xprize competition. The main components of the reaction are not included here and are given generic names such as enzyme X as they are currently not protected under IP. The steps outlined with tubes labeled with the generic names would allow any lab to reproduce the method. </div><div class = "text-block">Version 1 of this method outlines the protocol for a fluorescence-based readout that can be used with a qPCR machine or TECAN style plate reader. </div><div class = "text-block">Version 2 (to be released shortly) will be a colorimetric readout that can be assessed by eye. </div><div class = "text-block">The outlined protocol assumes the user performing this in a laboratory setting will setup  reactions in a clean room and analyze results in a seperate post-amplification room. In addition, since our amplification is rapid and at room temperature, care but be taken to add ligand to the master mix quickly and the reactions be sealed. </div></div>

[STEPS]
?. [Set-up]
Resuspend freeze dried master mix 1 with  of water
7.04 µl
?. [Set-up]
Add
0.3 µl
?. [Set-up]
Add Undisclosed enzyme Z
0.5 µl
?. [Set-up]
Add Undisclosed Enzyme Y
0.5 µl
?. [Set-up]
Add Undisclosed Enzyme X
0.13 µl
?. [Set-up]
Resuspend freeze dried master mix 2 in   water and add to above
1 µl
?. [Set-up]
Put mix in Add  of sample and seal plate.
0.5 µl
A large master mix of the above components can be mixed and aliquoted into wells.
?. [Data collection]
Place plate in TECAN plate reader. Set gain to 120, excitation 480 and emission 550. A qPCR machine can also be used. Collect data at T= 30 min.Data can also be collected every 1s for 30 min to monitor kinetics of reaction in the particular plate reader being used if not TECAN SPARK reader. Fluorescence values of >4000 are positive and
?. [Sample collection]
Sample should be collected 1 hour after any food is consumed.10 minutes before collection rinse mouth well with water. Collect passive drool or pooled salive below tongue into collection tube.
TITLE: Wholemount Edu Staining (Zebrafish Larvae)
AUTHORS: Henna Myllymaki, Abigail Elliot, Yi Feng

[DESCRIPTION]
Methods for evaluating cell proliferation are important for research in the field of developmental biology, cancer biology and cell biology. Recent years, Click-iT EdU Assays by Invitrogen is gaining popularity due to its fast staining speed, mild tissue treatment condition, reproducibility and specificity. However, the standard kit from Invitrogen is designed for tissue cultured cells, hence the manufactory provided protocol is optimized for cells cultured on coverslips. EdU staining became a popular method to evaluate cell proliferation in vivo in zebrafish embryos. However, the protocols that we have tested in the past often yield inconsistent results due to variable EdU incorporation efficiency. Here we describe an EdU staining protocol that we optimized for zebrafish larvae, which incorporated a crucial EdU injection step. Our protocol was used successfully by many new students in training, without any issues. 

[STEPS]
SECTION: EdU staining
1. Inject 2nl bolus of 500uM EdU into the yolk of embryos


 

SECTION: EdU staining
2. Incubate at 28.5°C in the dark for 2.5 hours (this can be between 2 -3 hours, but be consistent between experiments; we have done 2 hours)
SECTION: EdU staining
3. Fix in 4% PFA (30min, Room temperature)25 °C 
30 min 

SECTION: EdU staining
4. Wash in 0.1% PBT (5min, RT shaking)    

SECTION: EdU staining
5. Wash in 3% BSA, Dilute in PBT (5min, RT shaking) -grams/100ml, i.e. 3g/100ml, 300mg in 10ml.  

SECTION: EdU staining
6. Block in 3% BSA, Dilute in PBT (1h, shaking)  

SECTION: EdU staining
7. Incubate in staining solution (as per kit protocol) – we use 250ul per sample (10-15 larvae)  30 min,  
250 µL   

SECTION: Immunostaining in dark (EGFP or hRAS)
8. Wash in PBT (5min, RT shaking x3)   

SECTION: Immunostaining in dark (EGFP or hRAS)
9. Wash in 5% Goat Serum, Dilute in PBT (5min, RT shaking)  

SECTION: Immunostaining in dark (EGFP or hRAS)
10. Block in 5% Goat Serum, Dilute in PBT (at least 2h, RT shaking)  

SECTION: Immunostaining in dark (EGFP or hRAS)
11. Incubate with anti-GFP Primary Ab 1:200, dilute in 5% Goat Serum in PBT (O/N, +4 °C shaking)
  
250 µL 

 


SECTION: Immunostaining in dark (EGFP or hRAS)
12. Wash in PBT (25-30 min, RT shaking) x 8-10 à >4 hours in total!!!  

SECTION: Immunostaining in dark (EGFP or hRAS)
13. Incubate with Secondary Ab [] 1:250, Dilute in 3% Goat Serum in PBT (overnight at +4 °C -- prefered)  

 

SECTION: Immunostaining in dark (EGFP or hRAS)
14. Wash in PBT (15 min, RT shaking) x4  

SECTION: Immunostaining in dark (EGFP or hRAS)
15. Stain with Hoechst 33258 Dilute 1 in 1000 in PBT (30mins, RT shaking)  

 

SECTION: Immunostaining in dark (EGFP or hRAS)
16. Wash in PBT (15mins, PBT) x4  

SECTION: Embed and Image
18. Mounting with cover-slip and glass slides. 
SECTION: Embed and Image
17. Incubate in AF1 solution.
TITLE: Crystallization of SARS-CoV-2 nsp15 NendoU
AUTHORS: Peter Marples, Daren Fearon, Lizbé Koekemoer, Andre Schutzer de Godoy

[DESCRIPTION]
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). The NSP15 endoribonuclease enzyme, known as NendoU, is highly conserved and plays a critical role in the ability of the virus to evade the immune system. NendoU is a promising target for the development of new antiviral drugs. This protocol was used to grow SARS CoV-2 NSP15 crystals that were applied high-throughput crystallographic fragment screening at XChem.

[STEPS]
SECTION: Crystallization experiment
3. Protein and buffer requirements:
86.4 µL3.4 mg/mL  
2.88 mL 
2.88 µL   seeds, no dilution

SECTION: Crystallization experiment
5. Dispense 30 µL into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense 300 28613.4 mg/mL    to each lens using the SPT mosquito.
Dispense  290 2861 to each lens using the SPT mosquito.
Dispense  10 2861 to each lens using the SPT mosquito.

Drop ratio: 30:29:1 ratio (300 nl   : 290 nl reservoir solution: 10 nl seeds)
Final drop volume: 600 nl
SECTION: Crystallization experiment
4. Crystallisation screen composition:
0.1 Molarity (M)  sodium citrate pH 5 
14% w/v PEG 6000

Stock solutions used:
1 Molarity (M)  sodium citrate adjusted to pH 5  with HCl
50% w/v PEG 6000

 

SECTION: Crystallization experiment
7. Crystal form after ~96 h.
 


SECTION: Crystallization experiment
6. Incubate at 20 °C for 1440 min in Formulatrix Rock Imager.

Imaging Schedule: The first images are taken after 12 h and the imaging schedule follows a Fibonacci sequence of days for further collections. 
SECTION: Equipment needed
1. Formulatrix Rock Imager (or incubator of choice)
SPT mosquito
 
P100 8 multi-channel pipette
SwissCI 3 lens plate

SECTION: Crystallization experiment
2. Prepare seed stock:
 
No dilution    seeds
SECTION: Data collection at Synchrotron
8. Diamond Light Source
Unattended Data Collection (UDC)
Data Collection Temperature: 100K
Detector: DECTRIS EIGER2 X 9M
Beamline: I04-1
Wavelength:  0.9127 Å
Resolution (Å): 2.00
Beam Size (μm): 60 X 50
Number of images: 1500
Oscillation: 0.12°
Exposure (s): 0.0400
Transmission (%): 100
Flux (ph/s): 93.50e+11
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Contact Dr. Steven Wilhelm (wilhelm@utk.edu) or Josh Stough (<a target="_blank"><span title="jstough@vols.utk.edu">jstough@vols.utk.edu</span></a>) for additional information regarding this protocol.</p>

[BEFORE_START]
<p>You will need:</p>
<p>Assembled sequencing dataset--the dataset to be screened needs to be assembled and the contigs extracted. You may want to filter out contigs that are too short or have low coverage.</p>
<p> </p>
<p>Marker gene sequence list--to screen the dataset, you will need a file containing the sequences of the genes that you want to screen for, whether they are DNA or protein sequences.</p>

[STEPS]
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TITLE: Protocol-RNA-ISH
AUTHORS: Klaus Hirschbühl (Hematology and Oncology Medical Faculty University of Augsburg Augsburg Germany)

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:center">Protocol-RNA-ISH to the paper:</div></div><div class = "text-block"><div class = "justify" style = "text-align:center"></div></div><div class = "text-block"><div class = "justify" style = "text-align:center"><span style = "font-weight:bold;">Viral mapping in COVID-19 deceased in the Augsburg autopsy series of the first wave: a multiorgan and multimethodological approach</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:center"><span style = "font-weight:bold;">Full organ mapping of patients with COVID-19: an autopsy study</span></div></div></div>

[STEPS]
?.
TITLE: Crosslinking of Cultured Cells
AUTHORS: Eric L. Van Nostrand, Thai B. Nguyen, Chelsea Gelboin-Burkhart, Ruth Wang, Steven M. Blue, Gabriel A. Pratt, Ashley L. Louie, Gene W. Yeo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Profiling of RNA binding protein targets in vivo provides critical insights into the mechanistic roles they play in regulating RNA processing. The enhanced crosslinking and immunoprecipitation (eCLIP) methodology provides a framework for robust, reproducible identification of transcriptome-wide protein-RNA interactions, with dramatically improved efficiency over previous methods. Here we provide a step-by-step description of the eCLIP method, along with insights into optimal performance of critical steps in the protocol. In particular, we describe improvements to the adaptor strategy that enables single-end enhanced CLIP (seCLIP), which removes the requirement for paired-end sequencing of eCLIP libraries. Further, we describe the observation of contaminating RNA present in standard nitrocellulose membrane suppliers, and present options with significantly reduced contamination for sensitive applications. These notes further refine the eCLIP methodology, simplifying robust RNA binding protein studies for all users.</div></div>

[STEPS]
?. [Preparation of Cultured Cells for Crosslinking]
Aspirate the spent media and wash the plate with 1× DPBS (~ for a 15 cm plate) at . For suspension cells, pellet cells to remove spent media, and wash once with 1× DPBS.
15 mL
0 Room temperature
?. [Preparation of Cultured Cells for Crosslinking]
Aspirate the supernatant from previous step, and add enough 1× DPBS (~ for a 15 cm plate) to cover the plate. For suspension cells, resuspend in 1× DPBS to a cell density of 2 × 107cells per mL, place  of cell suspension in 1× DPBS on a 10 cm plate.
5 mL
3 mL
?. [Crosslinking of Cultured Cells]
Place the tissue culture plate  or cooling block, and place the entire apparatus into the UV crosslinker, making sure the plate is leveled. Remove lid prior to crosslinking.
on ice
?. [Crosslinking of Cultured Cells]
Crosslink the cells with an energy setting of 400 mJ/Cm2.
?. [Crosslinking of Cultured Cells]
While keeping the cells , use a cell scraper to scrape the plate. Transfer the cells to a 50 mL conical tube. Wash the plate with  and add the wash to the 50 mL conical tube. Gently resuspend the cell until a homogenous mixture is obtained.
on ice
[1× DPBS]
?. [Crosslinking of Cultured Cells]
Spin cells down at .
Centrifuge: 200 34, 5 min
?. [Crosslinking of Cultured Cells]
Aspirate the supernatant. Resuspend in the desired amount for flash freezing, typically 2 × 107cells per mL.
?. [Crosslinking of Cultured Cells]
Transfer desired amount into 1.5 mL epi-tubes, and then spin down at . Aspirate the supernatant and freeze by submerging the epi-tubes completely in liquid nitrogen. Store at .
Centrifuge: 200 34, 5 min
-80 °C
TITLE: No Title
AUTHORS: 

[BEFORE_START]
Prepare High salt buffer,&nbsp;Carbonate buffer,Wash buffer,Protease inhibitors<br />Instruments you need:&nbsp;ULTRA-TURRAX Dispenser T10 basis S25 (IKA)(or other dispensers);&nbsp;Ultracentrifuge.

[STEPS]
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TITLE: Fast Green/Sirius Red Protocol For Leica ST5020 Automated Stainer
AUTHORS: Angela Denn, Zbigniew Mikulski

[DESCRIPTION]
This Sirius Red Fast Green protocol is for the Leica ST5020 Automated multistainer. You can use it as a guide to developing your own protocol, catered to the needs of your researchers. This template can be modified for manual staining but thebest and most reproducible results are easier to achieve with an automated stainer. 

This fork describes modifications to obtain high-quality special stain demonstrating collagen in FFPE and frozen tissues. 

[GUIDELINES]
 Daily Maintenance for ST5020
 
 



 Weekly Maintenance for ST5020

 




 Monthly Maintenance for ST5020

 

Quarterly Maintenance for ST5020
Replace active carbon filter quarterly

 

 

[STEPS]
SECTION: Fast Green/Sirius Red Stain
2.  Arrange the reagents in the stainer according to the layout (note dark blue positions for FG/SR cups) 

 


SECTION: Fast Green/Sirius Red Stain
3. 
Program the FG/SR protocol into the ST5020 stainer according to the table below

 


SECTION: Fast Green/Sirius Red Stain
1. Perform all required maintenance on the stainer and prepare working solutions
SECTION: Expected results
4. Collagen fibers should be stained red and all other non-collagen proteins should be stained green.
TITLE: Fluorescent Western Protocol
AUTHORS: Steven Burgess

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Analysis of proteins using fluorescent immunoblot.  </div><div class = "text-block"><span style = "font-weight:bold;">Note: </span><span style = "font-weight:bold;"> </span></div><div class = "text-block">- The choice of secondary antibody depends on the choice of primary antibody, whether it is derived from a mouse (monoclonal) or a rabbit (polyclonal).  </div><div class = "text-block">- It is advisable to stick to the 800CW wavelength to avoid problems with chlorophyll autofluorescence encountered with the 680CW antibodies.  </div><div class = "text-block"><span style = "font-weight:bold;">Literature: </span><a href="https://www.licor.com/documents/fxc6evxvxbub4srkqy6i9yg46l7i0xz5" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.licor.com/documents/fxc6evxvxbub4srkqy6i9yg46l7i0xz5</span></a></div></div>

[STEPS]
?. Keep membranes in the black Western Blot incubation box for all steps, this is important after adding the secondary antibody because the signal is light-sensitive and will become bleached if not exposed to light for a long enough period.
?. Wet with 1x PBS for  min
?. Rinse membrane with dH2O
?. Discard PBS and incubate with   at
0 Room temperature
As an alternative to Intercept Blocking Buffer you can use PBS-T 5% w/v milk powder. This is to prevent unspecific binding of antibody and lowers background signal
?. Incubate with primary antibody (appropriate dilution in Intercept Blocking Buffer) at   (in cold room) with gentle agitation on a platform shaker  .
4 °C
4 33
?. Prepare  PBS-T solution
1 L
?. Pour off the primary antibody and rinse the membrane with PBS-T.
Some primary antibodies can be re-used multiple times depending on the concentration used, in this instance collect the primary antibody in a tube and store the solution at -20 oC before re-use.
?. Cover the membrane with PBS-T, shake vigorously on a platform shaker at . Repeat 3 times
33
?. Create a working dilution of secondary antibody using  with 0.2 % (v/v) Tween, 0.01 % SDS (w/v). IRE Dye secondary antibody (1:20,000).
is sufficient to cover one blot and 0.01% SDS is only to be used in conjunction with PVDF membranes.
?. Incubate for  with gentle agitation on a platform shaker.
0 Room temperature
?. Pour off the secondary antibody
?. Cover the membrane with PBS-T, agitate
33
?. Discard PBS-T. Repeat step 12  three times
Note: more washes (x5) and for longer can be done to reduce background
?. Rinse then cover the membrane with 1x PBS, . Repeat (2X)
33
?. Proceed to imaging blot on LI-COR Odyssey CLx imaging system
TITLE: Expression and purification of LRRK2 constructs
AUTHORS: Verena Dederer, Deep Chatterjee, Stefan Knapp, Sebastian Mathea

[DESCRIPTION]
Expression and purification of human LRRK2 and its variants from insect cells.

[BEFORE_START]
Buffers, beads, columns are chillled at 4°C prior to use.

[GUIDELINES]
if not otherwise mentioned every step was performed on ice.


[STEPS]
SECTION: Protein expression and purification
1. exponentially growing SF9 cells (2x10^6 cells/mL in Lonza Insect-XPRESS medium) are transduced with high-titre bacculovirus suspension (encoding TEV cleavable N-terminally His6-Z-tagged human LRRK2).
SECTION: Protein expression and purification
2. After shaking with  at27 °Cfor 3960 min, cells are harvested by centrifugation.

SECTION: Protein expression and purification
3. Pellets are washed once with PBS and frozen until further use.
SECTION: Protein expression and purification
4. Cell pellets are resuspended in lysis buffer and lysed by sonication on ice using a 13-mm probe (35% amplitude, 5 sec pulse, 10 sec pause, 5 min total pulse time).
SECTION: Protein expression and purification
5. Lysate is cleared by ultracentrifugation for 1 hour with 100.000 x g, 4 °C


SECTION: Protein expression and purification
6. Clarified lysate is loaded onto pre-equilibrated Ni-NTA in gravity flow columns.
SECTION: Protein expression and purification
7. After extensive washing with lysis buffer  (20 CV), the His-tagged protein is eluted with elution buffer containing 0.300 Molarity (M)

SECTION: Protein expression and purification
8. After dilution of the eluate's salt concentration to 250 millimolar (mM) using no salt buffer, the protein is loaded onto a SP sepharose column connected to an Äkta Start system (Cytiva).

SECTION: Protein expression and purification
9. After washing with buffer containing 250 millimolar (mM) the bound LRRK2 construct is eluted with a salt gradient ranging from 250 mM to 2.5 M NaCl while elution fractions are collected throughout the run.

SECTION: Protein expression and purification
10. Peak fractions are pooled and TEV protease (molar ratio of 1:100) is added during overnight incubation rolling at 4 °C.

SECTION: Protein expression and purification
11. Cleaved tag, TEV protease and other contaminating proteins are removed by an combined SP sephareose-Ni-NTA rebinding.
SECTION: Protein expression and purification
12. LRRK2 construct is concentrated and subjected to gel filtration in gel filtration buffer on a HiLoad 16/600 S200 column using an Äkta Pure system.
SECTION: Protein expression and purification
13. Peak fractions are combined, the protein is concentrated.
SECTION: Protein expression and purification
14. the UV absorbance is measure and the protein is flash frozen in liquid nitrogen for storage.