eamag/protocols-with-steps huggingface dataset:
TITLE: Crystallization of Zika NS5 RdRp
AUTHORS: anu.chandran, Peter Marples, Martin Walsh

[DESCRIPTION]
The main aim of this work was to identify small molecules that bind Zika NS5 RdRp (catalytic RNA-dependent RNA polymerase domain) through X-ray fragment-based screening. The Zika NS5 RDRP domain was cloned, expressed, purified, and crystallised.  Suitable crystals for fragment screening were produced and optimised allowing an extensive fragment campaign to be performed. A native high-resolution structure was determined at 1.8Å and formed the basis for the fragment campaign.  

[STEPS]
SECTION: Crystallization experiment
2. Protein and buffer requirements:
28.8 µL5 mg/mL  
3.264 mL 
SECTION: Crystallization experiment
4. Dispense 34 µL into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense 100 28615 mg/mL    to each lens using the SPT mosquito.
Dispense  50 2861 to each lens using the SPT mosquito.

Drop ratio: 2:1 ratio (100 nl   : 50 nl reservoir solution)
Final drop volume: 150 nl
SECTION: Crystallization experiment
3. Crystallisation screen composition:
Morpheous I E10 condition
 
0.12 Molarity (M) Ethylene glycols
0.1 Molarity (M) Buffer system 3 pH 8.5
30 % v/v Precipitant Mix 2
Stock solutions used:
Morpheous I E10 condition

 

SECTION: Crystallization experiment
6. Crystal form after ~24 h.
 


SECTION: Crystallization experiment
5. 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: Data collection at Synchrotron
7. Diamond Light Source
Unattended Data Collection (UDC)
Data Collection Temperature: 100K
Detector: DECTRIS EIGER2 X 9M
Beamline: I04-1
Wavelength:  0.9212 Å
Resolution (Å): 1.78
Beam Size (μm): 60 X 50
Number of images: 3600
Oscillation: 0.10°
Exposure (s): 0.0020
Transmission (%): 100
Flux (ph/s): 9.50e+11
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Young people in Ghana, like young people globally, need access to information and services to have safe, healthy sexual and reproductive lives. Young people in Ghana, though, face a number of challenges accessing the sexual and reproductive health information and services they need, as evidenced by high levels of unmet need for family planning, early pregnancy and childbirth, and unsafe abortion.</p>
<p> </p>
<p>This project aims to explore the internal and external determinants of contraception use and use of services that address early/teen pregnancy, unintended pregnancy, and unsafe abortion among youth in Accra. To do this, we plan to perform a cross-sectional survey with 250 sexually active females and 100 sexually active males aged 18-24 years living in Accra-Tema. If we are able to secure additional funding, we also plan to perform four focus group discussions (8-12 participants each) with sexually active females aged 18-24 years on determinants of reproductive health care utilization so as to get a more nuanced understanding of these issues and the survey findings. Participants of both study components will be recruited from 1-2 low- and middle-income communities in Accra-Tema. The findings of this study will help service providers to tailor programs to better meet the needs of young people in urban Ghana.</p>
<p> </p>
<p>This project is being conducted by Ibis Reproductive Health, the University of Ghana School of Public Health, and Marie Stopes International/Marie Stopes International Ghana (MSI/MSIG).</p>

[STEPS]
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<p><strong>Supplemental notes:</strong></p>
<p>1)      An A<sub>260</sub> may be determined on a UV spectrophotometer (usually a 1:100 dilution works well).</p>
<p>         1 A<sub>260</sub> unit of PBCV-1 routinely yields 1.5-2.5 X 10<sup>10</sup> PFU/ml of virus.</p>
<p>2)      For critical work, a second purification through sucrose gradients or a set of iodixanol gradients may be necessary.</p>

[STEPS]
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TITLE: Size Exclusion Chromatography
AUTHORS: Verena Dederer, Stefan Knapp, Sebastian Mathea

[DESCRIPTION]
Size-exclusion chromatography (SEC) is a method for separating proteins according to their size. To achieve this, a protein sample is applied to a column that is tightly packed with porous beads.
The size separation is determined by the thickness of the hydration shell of the proteins in a solvent. Small molecules can penetrate the pores, while large molecules cannot and are eluted first from the column. Smaller proteins follow, whereby the elution time is inversely proportional to the protein size. 
We used this method to investigate whether proteins form a stable complex. The individual complex partners have a characteristic elution profile. If there is a stable interaction, they are expected to co-elute after mixing. 

[STEPS]
SECTION: Sample Preparation
1. Mix the two purified interacting proteins in appropriate buffer with the appropriate ratio.

In our case:  LRRK2RCKW +   DARPin E11 with a ratio of 1:5 in 2 mL  


 
 
SECTION: Sample Analysis
2. 2 mL sample were subjected to a S200 gel filtration column combined with an ÄKTA XPress system in running buffer: 20 mM HEPES pH 7.4, 500 mM NaCl, 2.5 mM MgCl2, 20 µM GDP, 0.5 mM TCEP, 0.5%
glycerol

SECTION: Sample Analysis
4. Analyse collected fractions by SDS PAGE and Coomassie stain or immunoblot analysis.
SECTION: Sample Analysis
3. Collect flowthrough fractions (e.g. 3-mL)
TITLE: E gene amplification
AUTHORS: Bo Yi, Yi Chen, Xiao Ma, Haibin Wang, Rong Wang, Keqin Ding, Lei Xie, Dongliang Zhang, Shuli Jiao, Xuying Lao, Yi-Chen Chiang, Yanhua Su, Benhua Zhao, Guozhang Xu, Tianmu Chen

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

[STEPS]
?. The dengue virus strains obtained from cell culture were extracted by TGuide S32 magnetic bead method DNA/RNA extraction kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. and amplified by one step RNA PCR Kit (Code No: DRR057A) reagent of Bao Bioengineering (Dalian) Co., Ltd.
?. The full sequence of E gene amplified by primer sequence was D1 (E) F: CAA GAA CCG AAA CA / GT GGA TGT C; D1 (E) R: GGC TGA TCG AAT TCC ACA CAC, and the length of amplified fragment was 1849 bp.
?. Reaction conditions: reverse transcription at 50 °C 30 minutes, reverse transcription at 94°C 2 minutes, reverse transcription at 94°C 30 seconds, reverse transcription at 52°C 30 seconds, reverse transcription at 72°C 2 minutes, 40 cycles and extension at 72°C. The amplified product was 5 μL, and 1.5% agarose gel electrophoresis was used to confirm the reaction product according to Marker position.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol aims to amplify the PreS1/PreS2 region of the Hepatitis B genome. It can be obtained from a single PCR giving an amplified product of 479 bp. However genotype D stains have typically a deletion in this region of 33 nucleotides, giving an amplified product of 446 bp. Furthermore, larger deletions may occur in rare HBV genotype D strains and the amplicon size may be as short as 263 bp. Used for phylogenetic analysis, this region has been shown to better fit with the full-length phylogenetic information of the HBV DNA, than the PreC/C region. However it may give ambigous results with regards to recombinant strains.</p>

[STEPS]
TITLE: Protocol for Modified Standard Method 9260.B2 for the Isolation of Salmonella from Surface Water
AUTHORS: Autumn L Kraft, Manan   Sharma, Jonathan G Frye, James  E Wells, Abasiofiok  Mark Ibekwe, NARMS EWG  

[DESCRIPTION]
This protocol details the Modified Standard Method 9260.B2 for the recovery and isolation of Salmonella from 1 L of surface water.


[STEPS]
SECTION: Protocol
1. Prepare for filtration by assembling vacuum set up, including a Buchner flask (collection) that fits the Pall magnetic filter funnel and can hold 1 L of water. 
 

SECTION: Protocol
2. Prepare a decontamination station for magnetic filter funnel by filling 2 1 L beakers with 900 mL 70% EtOH. 
 


SECTION: Protocol
3. Place one 47-mm glass fiber filter into the bottom magnetic filter funnel (Forceps work well to gently pull one filter from stack and place into filter funnel) attached to Buchner funnel.
SECTION: Protocol
3.1. If needed use sterile forceps to gently 47-mm glass fiber filter into place to fit into bottom of the funnel.  

SECTION: Protocol
4. If needed, add Control strain to surface water sample by gently tipping vial over the opening. Close bottle tightly and  mix sample thoroughly before proceeding.
SECTION: Protocol
5. Measure 2 different sterile 0.5 g portions of the cellulose pool-filter fiber (Aqua Dew™), contained in sterile tubes, and suspend ONE of those portions in 15 mL of sterile deionized water.

SECTION: Protocol
6. Pre-load glass filter with the pool-filter fiber by applying vacuum and pouring the 15 mL water / pool-filter fiber suspension over the glass fiber filter.

SECTION: Protocol
7. Add the second 0.5 g portion of pool-filter fiber to the 1 L of sample water, close bottle tightly, and vigorously mix suspension.

SECTION: Protocol
8. Filter water sample by carefully pouring into 300 mL funnel and over pre-loaded 47 mm glass filter and allow vacuum to completely filter water sample.
SECTION: Protocol
8.1.   

SECTION: Protocol
9. If desired, use sterile deionized water to rinse off pool-filter fiber from sides of Pall filter funnel before turning off vacuum.
SECTION: Protocol
10. Carefully and aseptically, using sterilized forceps, transfer the 47 mm glass fiber filter (filter cake) from the filter funnel into a Whirl-Pak® sample bag.   
 


SECTION: Protocol
11. Add 25 mL of 1 X BPW to Whirl-Pak bag and massage until filter cake until broken up.

SECTION: Protocol
12. Decontaminate filter funnel pieces (2) individually by dropping them into first beaker of 70% EtOH. Allow for a minimum of 2 min contact time before transferring to second 70% EtOH bath. After a second 2-min soak, rinse filter funnel pieces with DeI water and allow to dry.

SECTION: Protocol
13. Incubate Whirl-Pak bag containing BPW-enriched filter cake for 18-24 h at 37 °C.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This is the correct protocol if you are using the C2987H&nbsp;cells. If you are using the&nbsp;C2987I cells, please refer to <a href="http://protocols.io/view/High-Efficiency-Transformation-Protocol-C2987I-imst35" target="_blank">this protocol</a>.

[GUIDELINES]
<strong>Transformation Protocol Variables</strong><br /><br /><strong>Thawing</strong>:&nbsp;Cells are best thawed on ice and DNA added as soon as the last bit of ice in the tube disappears. Cells can also be thawed by hand, but warming above 0&deg;C will decrease the transformation efficiency.<br /><br /><strong>Incubation of DNA with Cells on Ice</strong>:&nbsp;For maximum transformation efficiency, cells and DNA should be incubated together on ice for 30 minutes. Expect a 2-fold loss in transformation efficiency for every 10 minutes this step is shortened.<br /><br /><strong>Heat Shock</strong>:&nbsp;Both the temperature and the timing of the heat shock step are important and specific to the transformation volume and vessel. Using the transformation tube provided, 30 seconds at 42&deg;C is optimal.<br /><br /><strong>Outgrowth</strong>:&nbsp;Outgrowth at 37&deg;C for 1 hour is best for cell recovery and for expression of antibiotic resistance. Expect a 2-fold loss in transformation efficiency for every 15 minutes this step is shortened. SOC gives 2-fold higher transformation efficiency than LB medium; and incubation with shaking or rotating the tube gives 2-fold higher transformation efficiency than incubation without shaking.<br /><br /><strong>Plating</strong>:&nbsp;Selection plates can be used warm or cold, wet or dry without significantly affecting the transformation efficiency. However, warm, dry plates are easier to spread and allow for the most rapid colony formation.<br /><br /><strong>DNA Contaminants to Avoid</strong><br />
<table width="468">
<tbody>
<tr>
<td><strong>Contaminant</strong></td>
<td><strong>Removal Method</strong></td>
</tr>
<tr>
<td>Detergents</td>
<td>Ethanol precipitate</td>
</tr>
<tr>
<td>Phenol</td>
<td>Extract with chloroform and ethanol precipitate</td>
</tr>
<tr>
<td>Ethanol or Isopropanol</td>
<td>Dry pellet before resuspending</td>
</tr>
<tr>
<td>PEG*</td>
<td>Column purify or phenol/chloroform extract and ethanol precipitate</td>
</tr>
<tr>
<td>DNA binding proteins (e.g. Ligase)</td>
<td>Column purify or phenol/chloroform extract and ethanol precipitate</td>
</tr>
</tbody>
</table>
<br />* Ideally, DNA for transformation should be purified and resuspended in water or TE. However, up to 10 &micro;l of DNA directly from a ligation mix can be used with only a two-fold loss of transformation efficiency. Where it is necessary to maximize the number of transformants (e.g. a library), a purification step, either a spin column or phenol/chloroform extraction and ethanol precipitation should be added.

[STEPS]
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TITLE: Cell Counting
AUTHORS: Laura Sánchez

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Bacterial concentration can be measured by several methods. One of them is turbidity. Turbidity, or light scattering, is measured in a spectrophotometer such as a Spectronic 20. This method has the advantage of being the quickest method at the lab bench.</div></div>

[STEPS]
?. [Making an absorbance curve for Escherichia coli]
Turn on the spectrophotometer and set the wavelength to 425 nm. Let it warm up for at least 15 min.
?. [Making an absorbance curve for Escherichia coli]
Label the culture tubes 1 – 21. Using different pipets, add turbid E. coli culture and TSB to each tube in the volumes shown in the table.
?. [Making an absorbance curve for Escherichia coli]
Mix each tube thoroughly by vortexing for 1 – 2 sec with a vortex mixer.
?. [Making an absorbance curve for Escherichia coli]
With no tube in the spectrophotometer, set the 0%T setting.
?. [Making an absorbance curve for Escherichia coli]
Transfer 3 – 4 ml of broth from tube 21 (TSB with no E. coli) to the spectrophotometer cuvette, carefully wipe the outside of the tube with a kimwipe, place the tube in the machine, and set the 100%T setting (blank the spectrophotometer). Switch the spectrophotometer setting to “Absorbance.”
?. [Making an absorbance curve for Escherichia coli]
Remove the cuvette and pour the broth back into tube 21. Carefully drain the last few drops from the lip of the cuvette into a kimwipe. Put the contaminated kimwipe in the biohazard bag.
?. [Making an absorbance curve for Escherichia coli]
Starting with tube #20 and going backwards to tube #1, repeat steps 5-6.
?. [Making an absorbance curve for Escherichia coli]
Transfer the data to a Microsoft Excel spreadsheet. The spreadsheet should contain the following columns: volume of E. coli culture, volume of broth, dilution factor, and measured absorbance.
?. [Making an absorbance curve for Escherichia coli]
In the spreadsheet, create an X-Y scatter plot of dilution factor vs absorbance. * Please note: All charts must be added as separate sheets in the workbook, not pasted into the worksheet with the data columns.
?. [Making an absorbance curve for Escherichia coli]
Add a second order polynomial trendline to the plot, and set the options on the trendline to show the equation and the correlation coefficient.
?. [Making an absorbance curve for Escherichia coli]
From the plot, identify the range in which absorbance is proportional to bacterial concentration. Create a second plot, plotting only the linear range of dilution factor vs absorbance.
?. [Making an absorbance curve for Escherichia coli]
Add a linear trendline to the second plot, and set the options for the trendline to show the equation and the correlation coefficient.
TITLE: SH-SY5Y Transduced with HLA-A2 mCherry Lentivirus Sorting Protocol
AUTHORS: Ali Albalakhi, Ning Xia

[DESCRIPTION]
This is the cell sorting protocol.

[STEPS]
1. Aspirate the medium, wash with 2mL DPBS twice
2. Add 2mL Trypsin to the 60mm dishes and incubate for 2mins to lift the cells
3. Add 2ml complete medium to stop trypsinization, and pipette up and down to collect all cells
4. Transfer all cell suspension into a 15ml conical tube, spin down to get the cell pellet 200g for 4min
5. Resuspend each cell pellet in 1ml sorting medium (Add 2% (vol/vol) KnockOut serum replacement to 50 ml of DPBS. Can be stored at 4 °C for 6 weeks.) 

To make 50 mL add 1mL of KnockOut serum into 49mL DPBS
6. Prime the cell strainer with 2mL of sorting medium making sure to cover the entire mesh.
7. Discard the sorting medium in the 50mL tube
8. Apply each cell suspension to the center of a cell strainer (pushing through with pipette where necessary, and – with a new tip – pulling off strained cell suspension stuck to the bottom of filter).
9. After straining the cell suspension, add about 5µL of sorting medium to wash the strainer for any left-over cells.
10. Aliquot cell suspension into sorting tubes and put it on ice.
11. Add DAPI (diluted 1:10,000 to make final concentration at 0.1ug/ml) to the strained cell suspension. This helps to distinguish live from dead cells

0.1µL per 1mL 
SECTION: To prep for FACS: For each condition,
12. Take 2 culture tubes with 1 mL sushi expansion medium to catch the sorted cells 
Take 3ml extra sorting medium (in case they ask us to dilute the sample) put everything on ice to take to the FACS facility

SECTION: Sorting Parameters:
13. Go to the FACS facility at 149, 5th floor, and ring the bell to be let in.
SECTION: Sorting Parameters:
14. (i) Use nozzle 1 (100um)
(ii) mCherry detection (blue channel; ex: 587nm; em: 610)
(iii) Just collect mCherry-positive cells; give them the sushi medium-containing tubes to collect cells
(iv) Tell them you want to try to get >200,000 cells per condition where possible but prioritize getting through as many samples as possible.
(v) Can keep cold while sorting, or sort at RT (either is fine).
(vi) Can let them know how inclusive/restrictive to be when making gates. Threshold parameters include:
- sorting for singlets (cells on diagonal); doublets usually indicate 2 cells stuck together
- getting rid of particles that are likely debris.
- selecting the mCherry intensity threshold
TITLE: Prognostic value of combining 24-hour ASPECTS, and hemoglobin to red cell distribution width ratio on the THRIVE-score in predicting in-hospital mortality among ischemic stroke patients treated with intravenous thrombolysis.
AUTHORS: Sarawut Krongsut, Wipasiri Naraphong, Surachet Srikaew, Niyada Anusasnee

[DESCRIPTION]
Background:
Acute ischemic stroke (AIS) emerges as a significant global health issue, directly impacting mortality and disability. The Total Health Risk In Vascular Events (THRIVE) score, appreciated for its simplicity and ease of use, consistently validates its ability to accurately predict stroke clinical outcomes. However, the THIRVE score is currently lacking laboratory and neuroimaging data, which limits its ability to provide precise prediction of stroke outcomes. Our study evaluates the impact of integrating the 24-hour Alberta Stroke Program Early CT Score (ASPECTS) and hemoglobin-to-red cell distribution width (HB/RDW) ratio into the THRIVE score using the multivariable fractional polynomial (MFP) method (combined THRIVE-MFP) compared to the THRIVE-c model. We aim to assess their added value in predicting in-hospital mortality (IHM) prognosis.
Methods: A retrospective study from January 2015 to July 2022 examined consecutive AIS patients receiving intravenous thrombolysis. Data on THRIVE scores, 24-hour ASPECTS, and HB/RDW levels were collected upon admission. The model was constructed using logistic regression and the MFP method. The prognostic value was determined using the augmented area under the receiver operating characteristic (AuROC). Ischemic cerebral lesions within the middle cerebral artery territory were evaluated with non-contrast computed tomography (NCCT) following 24 hours of intravenous thrombolysis completion (24-hour ASPECTS).
Results:
Among a cohort of 345 patients diagnosed with AIS who received intravenous thrombolysis, 65 individuals (18.8%) experienced IHM. The combined THRIVE-MFP model was significantly superior to the THRIVE-c model (AuROC 0.980 vs. 0.876, P<0.001).
Conclusion:
The combined THRIVE-MFP model showed excellent predictive performance, offering potential for guiding treatment decisions and enhancing healthcare outcomes. Incorporating 24-hour ASPECTS on NCCT and HB/RDW proved valuable in IHM prediction, particularly for hospitals with limited access to advanced neuroimaging resources.

[STEPS]
1. Prognostic value of combining 24-hour ASPECTS, and hemoglobin to red cell
distribution width ratio on the THRIVE-score in predicting in-hospital
mortality among ischemic stroke patients treated with intravenous thrombolysis
TITLE: Immunofluorescence and live-cell Imaging
AUTHORS: Shenjie Wu, Nancy C. Hernandez Villegas, Iona Thomas-Wright, Richard Wade-Martins, schekman

[DESCRIPTION]
This protocol contains a detail description of how to perform immunostaining on two different cell types, U2OS and iPSCs cells. 
It also describes how to perform live-cell imaging procedure using a Zeiss LSM900 confocal microscope in a temperature-controlled environment.

[STEPS]
SECTION: Immunofluorescence of U2 cells
1. U2OS cells were washed once with PBS and immediately fixed by 4% EM-grade paraformaldehyde for 10 min at Room temperature

SECTION: Immunofluorescence of U2 cells
2. Cells were washed three times with PBS for 10 min each time.

SECTION: Immunofluorescence of U2 cells
3. Blocked and permeabilized for 30 min in permeabilization buffer (5% FBS and 0.1% saponin in PBS)
SECTION: Immunofluorescence of hiPSC dopamine neurons
9. Cells were fixed with 4% paraformaldehyde in PBS and 0.1% Triton-X was used for permablization 10 min 
SECTION: Immunofluorescence of U2 cells
4. Cells were then incubated with 1:100 dilution of primary antibodies at 4 °C 60 min

SECTION: Immunofluorescence of hiPSC dopamine neurons
10. Blocked in 10% normal donkey serum for 60 min Room temperature 

SECTION: Immunofluorescence of hiPSC dopamine neurons
11.  Cells were then incubated with 1:100 dilution of primary antibodies at 4 °C 60 min

SECTION: Immunofluorescence of U2 cells
5. Cells were washed three times with PBS for 10 min each time.
SECTION: Immunofluorescence of hiPSC dopamine neurons
12. Cells were washed three times with PBS for  10 min each time.
SECTION: Immunofluorescence of U2 cells
6. Cells were incubated with 1:500 dilution of fluorophore-conjugated secondary for 30 min at Room temperature

SECTION: Immunofluorescence of hiPSC dopamine neurons
13. Cells were incubated with 1:500 dilution of fluorophore-conjugated secondary for 30 min at Room temperature

SECTION: Immunofluorescence of hiPSC dopamine neurons
14. Prolong Gold with DAPI was used as mounting solution
SECTION: Immunofluorescence of hiPSC dopamine neurons
15. Images were acquired with a Zeiss LSM900 confocal microscope and analyzed with Fiji/ImageJ software
SECTION: Immunofluorescence of U2 cells
7. Prolong Gold with DAPI was used as mounting solution
SECTION: Immunofluorescence of U2 cells
8. Images were acquired with a Zeiss LSM900 confocal microscope and analyzed with Fiji/ImageJ software
SECTION: Live-cell imaging
16. Cells were cultured in 35 mm glass bottom dishes (MatTek). 
SECTION: Live-cell imaging
17. HaloTag fluorescent ligands were added according to the manufacturer’s protocol (Promega). 
SECTION: Live-cell imaging
18. After incubation for 15 min in the incubator (37 °C and 5% CO2 ), the cells were quickly washed twice with PBS. The medium was replaced with Opti-MEM supplemented with 10% FBS. 

SECTION: Live-cell imaging
19. Imaging was performed using a Zeiss LSM900 confocal microscope in a temperature-controlled
(37 °C and 5% CO2) environment.