eamag/protocols-with-steps huggingface dataset:
TITLE: RNA sequencing analysis of transcriptomic responses to vagal nerve stimulation in myenteric ganglia of porcine colon
AUTHORS: Tao Li, Pu-Qing Yuan

[DESCRIPTION]
The region-dependent molecular characterization and influences by vagus nerve stimulation (VNS) influences in the porcine ENS has been little investigated so far. We use laser-capture microdissection coupled with bulk RNA sequencing analysis to profile the transcriptomes of myenteric ganglia (MG) from porcine proximal and distal colon and to evaluate discrepancy of the region-specific gene programs in MG after electrical stimulation of the celiac branch of the abdominal vagus nerve in anesthetized porcine.

[STEPS]
1. Six castrated male Yucatan minipigs (3 of each controls and 3 with VNS) were fasted for 12 h and anaesthetized. Those with VNS underwent electrical stimulation of the celiac branch of the abdominal vagus nerve (2 Hz, 0.3-4 ms, 5 mA, 10 min) using pulse train. 
2. The proximal and distal colonic (pC, dC) pecimens with full thickness were collected. All samples were embedded in OCT, snap-frozen in dry ice and stored at -80°C for LCM procedures. A range of 25-40 ganglia/subject were harvested from MG respectively of pC and dC (3 of each control and with VNS) using LMD-6000 Laser Micro-dissection System. Total RNA was extracted for construction of cDNA libraries and sequenced on an Illumina HiSeq 3000 sequencer as 50 base pair single-end reads.
3. All of the reads were then aligned to the Sus_scrofa genome using STAR. The differentially expressed gene (DEG) lists were generated using edgeR in conjunction with Limma-Voom. An FDR–adjusted p value threshold of q < 0.05 was used to subset meaningful genes, which were used for pathway enrichment analysis. Enrichment analysis for biological processes (BPs) was performed using g:Profiler.
4. The results from g:Profiler were inputted into EnrichmentMap in Cytoscape to visualize the networks of BPs. All annotations of enriched BPs with an FDR p-value cut-off of 0.05.
5. WikiPathways of interest were searched from human database and all genes involved in the pathways were downloaded. The genes were matched to the porcine DEG lists that were already matched to the generated high-quality human-porcine ortholog gene list. The resulting WikiPathways-matching DEG lists were searched in the grouped networks of BPs and all terms (BPs) containing the DEG were highlighted.
6. The significance (corrected p-value by Benjamini-Hochberg) of the highlighted terms were exported to compute the FDR p-value of the specified WikiPathways using R/harmonicmeanp package. Similarity matrix was generated in R/rrvgo package at default setting using DEG lists from comparison of p-pC-MG and p-pC-ISG in porcine with VNS.
TITLE: Ligation and gel electrophoresis
AUTHORS: Chia-Hsien  Shih

[DESCRIPTION]
This protocol is used for ligation of circular probe

[STEPS]
SECTION: Preparation
1. Dilute linear DNA into 10µM

SECTION: Preparation
2. Dilute T4 ligation primer into 10µM
SECTION: Protocol of Ligation
3. Add 11 µL of RNase-free water into a eppendorf

SECTION: Protocol of Ligation
4. Add 3 µL of 10X T4 Ligase buffer 

SECTION: Protocol of Ligation
5. Add 9 µL of 10µM T4 ligation primer

SECTION: Protocol of Ligation
6. Add 3 µL of 10µM linear DNA

SECTION: Protocol of Ligation
7. Add 4 µL of T4 Ligase 

SECTION: Protocol of Ligation
8. Spin down after vortex
SECTION: Protocol of Ligation
9. Incubate for 270 min at 26 Room temperature

SECTION: Protocol of Gel Electrophoresis
10. Put 2% agarose gel into the electrophoresis tank.
SECTION: Protocol of Gel Electrophoresis
11. Prepare 0.5X TAE buffer ( add 3.5 mL 50X TAE buffer and 350 mL ddH2O and mix them), then pour into the electrophoresis tank. Make sure the gel is soaked into the buffer completely.

SECTION: Protocol of Gel Electrophoresis
12. Take 5 µL of marker and load it in the first well.

SECTION: Protocol of Gel Electrophoresis
13. Take 1 µL of 6X loading dye and drop on the parafilm separately, pipetting dye with 5 µL of sample

SECTION: Protocol of Gel Electrophoresis
14. Load samples into each well
SECTION: Protocol of Gel Electrophoresis
15. Run gel at a constant voltage of 100V for30 min at room
temperature
SECTION: Protocol of Gel Electrophoresis
16. After gel electrophoresis, put the gel into gel reading machine and report the result.
TITLE: Cortical, Striatal and Dopaminergic Neurons Cultures Protocol
AUTHORS: giulia.tombesi

[DESCRIPTION]
This protocol describes how to obtain primary neuronal cultures from postnatal mice (P0-P1). It can be used to obtain pure cortical neurons cultures or stratal-cortical neurons and dopaminergic-cortical neurons co-cultures. 

[BEFORE_START]
Prepare the plates:

- Coat plates with poly-L-Lysine 10 min at room temperature or at least 120 min at 37°C.
- Wash thoroughly with distilled water three times. Let it dry under the hood in the dark.


Prepare the following solutions: 

- Dissection medium: 10mM HEPES pH7.3 , 0.1% D-glucose, 0.11mg/ml Sodium pyruvate in HBSS.
- DNase solution: one vial of DNAse in 500 µL of BME.
- Papain solution: one vial of Papain in 5 mL of BME plus 250 µL of DNase solution.
- Trypsin inhibitor solution: 15.5mg/ml in BME. Filter the solution.
- Stop solution: 250 µL of DNase solution, 600 µL of Trypsin inhibitor solution in 5.4 mL of BME.
- 10/10 solution: Trypsin inhibitor 10ug/ml and BSA 10ug/ml in BME. Filter the solution.  
- Plating medium: 1 mL B27 supplement, 125 µL L-Glutamine, 500 µL Pen/Strep, 500 µL N2 supplement in 50 mL Neurobasal. 


[STEPS]
SECTION: Cells Dissociation
2. Snip the tissues into smaller pieces and place them in Papain solution. Use around 500 µL of Papain solution for 5 SN, around 1 mL of Papain solution for 5 striata and around 5 mL of Papain solution for 5-6 cortices. 

SECTION: Cells Dissociation
3. Triturate 10 times with a 5ml-pipette 
SECTION: Cells Dissociation
4. Incubate at 37°C for 40 min and mix by inversion every 10 min
SECTION: Cells Dissociation
5. Triturate 10 times again with a 5ml-pipette
SECTION: Cells Dissociation
6. Spin in swing bucket rotor at 1000rpm for 5 min

SECTION: Dissection
1. Dissect the cortices, the striatum and the substantia nigra (SN) in cold dissection medium, carefully removing the meninges 
SECTION: Cells Dissociation
7. Remove and discard the supernatant
SECTION: Cells Dissociation
8. Tap the tubes to move the pellet and add STOP solution. Add 1 mL for 5 SN, 1 mL for 5 striata and around 3 mL for 3 cortices. 

SECTION: Cells Dissociation
9. Triturate 3 times with a 5ml-pipette
SECTION: Cells Dissociation
10. Incubate at room temperature for 10 min

SECTION: Cells Dissociation
11. Take the supernatant and add to 10/10 solution drop-by-drop (use a volume of 10/10 solution that is equal to the supernatant volume)  
SECTION: Cells Dissociation
12. Spin at 800rpm for 10 min

SECTION: Neuron Seeding
13. Discard the supernatant and resuspend in Neurobasal complete medium with AraC. Use 1 mL of medium to resuspend 5 striata and 3 cortices. 

SECTION: Neuron Seeding
14. Count cortical and striatal cells with trypan blue
SECTION: Neuron Seeding
15. Plate 500000 cells/well in 24wells-plate. 
For striatal-cortical co-culture plate a ratio of 1:2 striatum:cortex. 
For SN-cortical co-culture plate one well for each SN and around 500000 cortical neurons/well and also add GDNF to the medium. 
SECTION: Neuron Seeding
16. Replace half medium about every 7 days by collecting conditioned medium from cells and mixing with fresh complete medium in a ratio of 1:1.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>Intended use:</strong></p>
<p>Kinetic system for Aspartate Aminotransferase (AST) or Glutamic oxalacetic trasminase (GOT) determination.</p>
<p><strong>Teste Principle:</strong></p>
<p><strong>AST</strong> catalyzes specifically  the transfer of amino groups of asparctic acid to a Ketoglutarate yielding oxalacetate and Glutamate. The oxalacetate is reduced to malate  by the enzyme malate dehydrogenase (MDH) and  coenzyme NADH is oxidized NAD.</p>
<p>The reduction of the absorbance at 340 nm, as consequence of NADH oxidation, is determined photometrically and is direct proportional to the AST activity in the sample.</p>
<p>Aminotransferase determination involves the following reactions:</p>
<p>L- Aspartate + Ketoglutarate ---(AST) → Oxalacetate + L-Glutamate</p>
<p>Oxalacetate  + NADH ---(MDH)→  Malate + NAD</p>

[BEFORE_START]
<p>Spectrophotometer.</p>
<p>- Micropipettes and pipettes for measuring the stated volumes</p>
<p>- Water bath at temperature indicated under PROCEDURE.</p>
<p>- Stopwatch.</p>

[GUIDELINES]
<p>Reagents are for “in vitro” diagnostic use. Use the reagents according to the working procedures for clinical laboratories.</p>
<p>Storage and stability - Unopened reagents, when stored at indicated temperature, are stable up to expiration date shown on the label.</p>

[STEPS]
?. 
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TITLE: Wet Chemistry RNA Isolation Method for Mature Cassava Leaf Tissue
AUTHORS: Lynn   Doran

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

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

[STEPS]
TITLE: Study Medications (Part 5 of Safety and Efficacy of Imatinib for Preserving Beta-Cell Function in New-onset Type 1 Diabetes Mellitus)
AUTHORS: Stephen.Gitelman , Jeffrey A. Bluestone

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is Part 5 of "Safety and Efficacy of Imatinib for Preserving Beta-Cell Function in New-Onset Type 1 Diabetes Mellitus".</div><div class = "text-block">This clinical study is supported by JDRF. The aim of the collection is to determine whether imatinib will slow the progression of the autoimmune destruction of ß cells and lead to the preservation of C-peptide secretion in T1DM and to assess Diabetes-related objectives and safety of Imatinib in new-onset type 1 diabetes mellitus".</div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Protocol for cell separation, enumeration, and sorting of low biomass samples for SIP-NanoSIMS preparations. </p>

[BEFORE_START]
<ol>
<li>Clean 12 spoons
<ol>
<li>soaked in bleach ~15min, then rinse with 0.2um MQ, then Sonicate 15 min, then rinse, then bring water cup with spoons into clean bench and lay out on rnase away clean foil to dry. Small end of spoon is used.</li>
</ol>
</li>
<li>12 4% PFA/PBS 50 ml tubes with labels (8 ml added)</li>
<li>5 ml pipet and tips</li>
<li>1 ml pipet and tips</li>
<li>12 1.5 ml eppitubes with labels for IC</li>
<li>50 ml tube of glycerol solution</li>
<li>50 tube holders</li>
<li>screw driver to open stuck caps</li>
<li>Turn on static remover</li>
<li>Wash hands in front of static remover</li>
<li>Clean sample bottles with rnaseaway and place on clean foil</li>
</ol>

[STEPS]
?. 
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TITLE: Modified pH drop protocol (K+)
AUTHORS: Elizabeth Fozo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Modified pH drop protocol (K</span><span style = "vertical-align:super;">+</span><span style = "vertical-align:super;">)</span></div></div>

[STEPS]
?. [To prepare the K+ electrode for use:]
Remove the rubber caps.
?. [Do slope check for electrode performance:]
?. [Standard curve:]
0mM (no K+) = 9mL water + 1mL 1M NaCl
?. [Modified pH drop protocol:]
Read pH & mV readings atdesired intervals.
?. [To prepare the K+ electrode for use:]
Fill with 0.1M NaCl filling solution provided to just below the hole in the probe.
?. [To prepare the K+ electrode for use:]
Gently shake the probe like a thermometer to remove all air bubbles.
?. [To prepare the K+ electrode for use:]
Immerse the electrode in 0.1M KCl for 20-30 minutes.
?. [To prepare the K+ electrode for use:]
Rinse off.
?. [Do slope check for electrode performance:]
Lower the K+electrode into the beaker.
?. [Do slope check for electrode performance:]
Add 1mL 0.1M KCl to the beaker & keep stirring.  Once the reading is stable, record the mV reading (mine was-49.2).
?. [Do slope check for electrode performance:]
Add 10mL 0.1M KCl to the beaker.  Continue stirring.  Record mV reading (mine was6.1).
?. [Do slope check for electrode performance:]
The difference between the readings should be 52mV ± 2. (Mine was 55.3mV – called that good enough!)
?. [Standard curve:]
Take 1M stock KCl & dilute 1:10 in 8mL water + 1mL 1M NaCl to create the following K+ concentrations:0.1M (10-1) 0.01M (10-2)1mM (10-3)0.1mM (10-4)0.01mM (10-5),1µM (10-6)
?. [Standard curve:]
Read mV for each standard.  Create standard curve:  electrode potential (mV) vs. K+concentration (mM)
?. [Modified pH drop protocol:]
Prepare 100mL ON of culture as per pH drop protocol (in BHI + 1% glucose).
?. [Modified pH drop protocol:]
Measure OD700.
?. [Modified pH drop protocol:]
Pellet cells.
?. [Modified pH drop protocol:]
Wash cell pellet 2X in 30-35mL 1mM MgCl2.
?. [Modified pH drop protocol:]
Resuspend pellet in x mL 1mM MgCl2, where x= # from formula
?. [Modified pH drop protocol:]
For each experiment, take 4.5 mL cells + 0.5mL 1M NaCl → 0.1M NaCl in the final 5mL cell suspension.
?. [Modified pH drop protocol:]
Adjust pH to 7.2 with 0.5M NaOH.
?. [Modified pH drop protocol:]
Add 100µL 50% glucose = time zero.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Basic Protocol for culturing Neuro2a cells.

[GUIDELINES]
<strong>The following are taken directly from the vendor website, I use high glucose DMEM instead of Eagle's MEM. </strong><br /><br /><strong>Complete Growth Medium</strong> The base medium for this cell line is ATCC-formulated Eagle's Minimum Essential Medium, Catalog No. 30-2003. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 10%.<br /><br /><strong>Subculturing Volumes</strong> are given for a 75 cm2 flask. Increase or decrease the amount of dissociation medium needed proportionally for culture vessels of other sizes.&nbsp;Corning&reg; T-75 flasks (catalog #430641) are recommended for subculturing this product.

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

[DESCRIPTION]
This tutorial was based off of the paper The microbes we eat: abundance and taxonomy of microbes consumed in a day's worth of meals for three diet types. <span style="font-weight: 400;">The article provides those interested in bioinformatics, food science, or microbiology information about how to test the abundance and taxonomy of microbes in our daily meals. The step by step procedures covers how the author run this experiment. </span><br /><br /><span class="self-citation-authors">Source:<br />Lang JM, Eisen JA, Zivkovic AM.</span> (<span class="self-citation-year">2014</span>) <span class="self-citation-title">The microbes we eat: abundance and taxonomy of microbes consumed in a day’s worth of meals for three diet types</span>. <span class="self-citation-journal">PeerJ</span> <span class="self-citation-volume">2</span>:<span class="self-citation-elocation">e659. https://peerj.com/articles/659/</span>

[BEFORE_START]
Make sure you have QIIME installed.

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
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?.
TITLE: Immunohistochemistry for FOXP3+ staining in Breast Cancer Tissue
AUTHORS: Freda Halim

[DESCRIPTION]
These are protocols used for study of FOXP3+ Cell Count in Luminal B Her-2 negative BC patients. We used using paraffin sections of 66 samples and stained the slides for FOXP3+ antibody, using primary antibody clone 236A/E7 Abcam Cambridge UK dilution 1:50

[STEPS]
SECTION: Deparaffinization and Rehydration
1. Incubate slides in Xylenes for 3 minutes                                                              
SECTION: Deparaffinization and Rehydration
2. Incubate slides in Xylenes    
SECTION: Deparaffinization and Rehydration
3. Incubate slides in Xylenes  
SECTION: Deparaffinization and Rehydration
4. Rehydrate slides in 100% Ethanol
SECTION: Deparaffinization and Rehydration
5. Rehydrate slides in 96% Ethanol
SECTION: Deparaffinization and Rehydration
6. Rehydrate slides in 70% Ethanol
SECTION: Deparaffinization and Rehydration
7. Rinse with running water and aquadest
SECTION: Blockage of Endogenous Peroxidase
8. Incubate slides in 3% H202 
SECTION: Blockage of Endogenous Peroxidase
9. Rinse slides with running tap water and aquadest 
SECTION: Antigen Retrieval
10. Antigen Retrieval with Tris EDTA ( pH9) with pressure cooker, in 950 Celcius temperature
SECTION: Antigen Retrieval
11. Open the lid and cool down in room temperature
SECTION: Antigen Retrieval
12. Rinse slides with running water and aquadest 
SECTION: Antigen Retrieval
13. Rinse in PBS ( Phosphate Buffer Saline ) in pH 7.40-7.60
SECTION: Antigen Retrieval
14. Excell Block
SECTION: Antigen Retrieval
15. Rinse in PBS in pH 7.40-7.60
SECTION: Primary Antibody
16. Wipe excess liquid around the tissue
SECTION: Primary Antibody
17. apply primary antibody ( Clone 236A/E7 Abcam Cambridge UK dilution 1:50 ) 120μL
SECTION: Primary Antibody
18. Incubate for 60 minutes
SECTION: Primary Antibody
19. Rinse with PBS
SECTION: Secondary Antibody
20. Apply Excell Link as secondary antibody
SECTION: Secondary Antibody
21. Rinse with PBS
SECTION: Secondary Antibody
22. Apply Excell HRP as secondary antibody
SECTION: Signal Detection/Histochemistry
23. Apply DAB ( Diamino-benzidine ) 80-100μL for 10 minutes , Rinse with running tap water and aquadest for 5 minutes
SECTION: Signal Detection/Histochemistry
24. Apply Hematoxylline for 1 minutes, Rinse with running tap water and aquadest for 5 minutes
SECTION: Signal Detection/Histochemistry
25. Apply Tatcha's bluing solution and rinse with running tap water and aquadest for 5 minutes
SECTION: Dehydration and Clearing
26. Clear excess water from the slides
SECTION: Dehydration and Clearing
27. Dehydrate Slides in 70%, 96%, 100% for 5 minutes each
SECTION: Dehydration and Clearing
28. Incubate slides in Xylenes for 5 minutes
SECTION: Dehydration and Clearing
29. Mount the slides
TITLE: LCMS-SOP Determination of Microcystins in Water Samples by High Performance Liquid Chromatography (HPLC) with Single Quadrupole Mass Spectrometry (MS)
AUTHORS: Gregory L Boyer

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This standard operating procedure is used to determine and quantify microcystins and nodularin (intracellular and/or extracellular) in whole water samples or filter samples using HPLC-coupled with single quadrupole mass spectrometry (LC-MS). The method described here is a modification of the methods described in Boyer (2007) for particulate microcystins in water samples and does not use solid phase extraction. It was designed to use a 50% acidified methanolic extraction solvent, but is compatible with protocols for direct water analysis (US-EPA, 2016) and higher concentration methanolic extracts (Cerasino et al., 2017). It is not suitable for use with tissues samples or other samples with complex matrix interferences, such as soil or sediments. </div><div class = "text-block">The method is designed for high throughput (1000-1500 injections per month) using a low-resolution (unit mass) mass spectrometer. It gives results of similar accuracy to the commonly used Abraxis ADDA ELISA with additional information on the microcystin congener profiles. </div></div>

[STEPS]
TITLE: Overall protocol for top-down CZE-MS/MS of human small intestine tissue
AUTHORS: Jeannie  Camarillo, Bryon  Drown, Neil  Kelleher

[DESCRIPTION]
Overview description of the process for acquiring top-down capillary zone electrophoresis MS/MS data on human small intestine tissue

[STEPS]
SECTION: Tissue Collection
1. Small intestine tissue was provided by Stanford University TMC. Tissue was collected according to the following: 

https://dx.doi.org/10.17504/protocols.io.bin8kdhw
SECTION: Sample Preparation
2. 
 

SECTION: Data Acquisition
3. 
 

SECTION: Data Analysis
4.
TITLE: NEBNext® Ultra™ II End Repair/dA-Tailing Module (NEB #E7546)
AUTHORS: Menna Teffera, New England  Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The NEBNext Ultra II End Repair/dA-Tailing Module is optimized to convert 500 pg-1 μg of fragmented DNA to repaired DNA having 5 ́ phosphorylated, 3 ́ dA-tailed ends. </div><div class = "text-block">This module is part of the Ultra™ II workflow, and is optimized for use with the NEBNext™ Ultra II Ligation Module (NEB #E7595), for Illumina®-compatible library construction. </div><div class = "text-block">
This module is also compatible with some Oxford Nanopore MinION™workflows.</div><div class = "text-block">This module is designed for use with NEBNext Singleplex or Multiplex Oligos for Illumina (NEB #E7350, #E7335, #E7500, #E7600 or #E7535), NEBNext Ultra II Ligation Module (NEB #E7595), and NEBNext Ultra II Q5 Master Mix (NEB #M0544). </div><div class = "text-block"><span>Kits that include reagents for every step in the Ultra II DNA library construction workflow are also available (NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB </span><a href="https://www.neb.com/products/e7645-nebnext-ultra-ii-dna-library-prep-kit-for-illumina" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">#E7645</span></a><span>) and NEBNext Ultra II DNA Library Prep with Sample Purification Beads (NEB</span><a href="https://www.neb.com/products/e7103-nebnext-ultra-ii-dna-library-prep-with-sample-purification-beads" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;"> #E7103</span></a><span>).</span></div></div>

[STEPS]
?. [NEBNext End Prep]
Mix the following contents in a sterile nuclease-free tube:  AB1ComponentVolume2(green) NEBNext Ultra II End Prep Enzyme Mix3 µl3(green) NEBNext Ultra II End Prep Reaction Buffer7 µl4Fragmented DNA50 µl5Total Volume60 µl
AB1ComponentVolume2(green) NEBNext Ultra II End Prep Enzyme Mix3 µl3(green) NEBNext Ultra II End Prep Reaction Buffer7 µl4Fragmented DNA50 µl5Total Volume60 µl
?. [NEBNext End Prep]
Set a 100 μl or 200 μl pipette to 50 μl and then gently pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube.
Note: It is important to mix well. The presence of a small amount of bubbles will not interfere with performance.
?. [NEBNext End Prep]
Place in a thermocycler, with the heated lid set to ≥ 75°C, and run the following program:  at  at Hold at
20 °C
65 °C
4 °C
Safe Stop Point: If necessary, samples can be stored at –20°C; however, a slight loss in yield (~20%) may be observed. We recommend continuing with adaptor ligation before stopping.
?. [NEBNext End Prep]
Proceed directly to NEBNext Ultra II Ligation Module NEB #E7595.
TITLE: Poliovirus direct detection and nanopore sequencing (DDNS) FAQs
AUTHORS: Alex   Shaw, Catherine  Troman, Joyce Akello, Erika Bujaki, Manasi Majumdar, Javier Martin, Nick  Grassly

[DESCRIPTION]
This short FAQ document summarises the poliovirus direct detection and nanopore sequencing (DDNS) protocol, the equipment and reagents required, the cost and staff-time involved (Feb 2022).

[STEPS]
TITLE: Fixative preparation for Baermann funnel extraction
AUTHORS: Casey A Schlenker

[DESCRIPTION]
Fixative preparation for Baermann funnel extraction

[STEPS]
SECTION: Fixative Preparation
1. Weigh 1.6 g PFA powder

SECTION: Fixative Preparation
2. Dissolve in 20 mL of DI water

SECTION: Fixative Preparation
3. Incubate in water bath at 60 °C to help dissolve PFA powder for 40 min

SECTION: Fixative Preparation
4. Add 20 µL of 1 M NaOH to further help dissolve PFA powder

SECTION: Fixative Preparation
5. Place back in 60 °C water bath for 10 min

SECTION: Fixative Preparation
6. Pull out of water bath and cool to Room temperature

SECTION: Fixative Preparation
7. Add two tablets of PBS to a 100 mL volumetric flask

SECTION: Fixative Preparation
8. Mix 20 mL of each solution to get 4% formaldehyde

SECTION: Fixative Preparation
9. Add 400 µL of glycerol to fixative

SECTION: Fixative Preparation
10. Ensure pH 7.5

SECTION: Fixative Preparation
11. Place fixative in a hot water bath till 60 °C

SECTION: Fixative Preparation
5.1. If still not dissolved, add 10 µL more of 1M NaOH