eamag/protocols-with-steps huggingface dataset:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>These protocols accompany the following <em>GigaScience</em> publication:</p>
<p> </p>
<p>Benjamin Istace, et al. (2017) De novo assembly and population genomic survey of natural yeast isolates with the Oxford Nanopore MinION sequencer. <em>GigaScience...</em></p>

[STEPS]
TITLE: 4: User-friendly protocol: Cost-efficient Primer Exchange Reaction (PER) concatemerization (SABER-FISH)
AUTHORS: Jocelyn Y. Kishi, Sylvain W. Lapan, Brian J Beliveau, Emma R. West, Allen Zhu, Hiroshi M. Sasaki, Sinem Saka, Yu Wang, Constance L Cepko, Peng Yin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol explains the cost-efficient Primer Exchange Reaction (PER) concatemerization.</div></div>

[STEPS]
?. [PER mix and extension]
Add mix to strip tube with  and mix.
[5µM hairpin]
?. [PER mix and extension]
Incubate for  at , then pause cycler.
37 °C
?. [PER mix and extension]
Remove tube from the cycler, add  and mix.
[10µM probe oligos]
?. [PER mix and extension]
Incubate at  for desired extension time (see Note 2).
37 °C
?. [PER mix and extension]
Mix with the following components :  AB1ComponentVolume ( µL)210×PBS103100mM MgSO4* (NEB)104dNTP mix* (A,C,T only 6mM each, NEB)55Clean.G (1µM)106Bst LF polymerase* (McLab)0.57H2O44.5
on ice
Add the polymerase last.
AB1ComponentVolume ( µL)210×PBS103100mM MgSO4* (NEB)104dNTP mix* (A,C,T only 6mM each, NEB)55Clean.G (1µM)106Bst LF polymerase* (McLab)0.57H2O44.5
?. [PER mix and extension]
Heat to  to inactivate the Bst polymerase.
80 °C
?. [PER mix and extension]
Cool to .
4 °C
?. [Check Extension lengths]
To check the lengths of extensions, load  with loading dye on a dense agarose gel (~). You can also load samples into   (Thermo Fisher G402001).
[of reaction]
[E-Gel EX agarose gels]
Note 5: We recommend using primary probes extended to ~500-650 nt and branch probes extended to ~250-450 nt.
Note 6: Because there aren’t G bases in the concatemer sequences, and they are highly single-stranded, intercalating dyes are not as effective as they are at staining other types of sequences (we recommend using the Sybr Gold dye). This means the bands may look faint on the gel but still be efficiently elongated, so don’t be surprised if you need to turn up the contrast quite a bit.
?. [Purification of probes]
While purification of probes is not usually required for cell applications, we have found it helpful for SABER-FISH in tissue.
?. [Purification of probes]
Purify probes by running  (for 500 nt probe) over one MinElute PCR purification column (Qiagen #28004). Add PB buffer to 7×the volume of reaction being purified.
[of reaction]
?. [Purification of probes]
Elute in .
[ddH2O]
?. [Purification of probes]
Determine concentration after purification by nanodrop using ssDNA setting.
?. [Purification of probes]
Store probes at .
-20 °C
TITLE: Maintenance of TissueCyte System
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocols provides detailed instructions for procedures needed for maintenance of the TissueCyte scanning systems.</div><div class = "text-block"><span style = "font-weight:bold;">Note</span><span>: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.</span></div></div>

[STEPS]
TITLE: Experiment 4
AUTHORS: Dikla Perez , Yael Steinhart , Amir Grinstein , Meike Morren 

[DESCRIPTION]
This experiment provides evidence for this effect in a field setting. We approached residents of a specific city (referred to in what follows as “Smallville”) and offered them the opportunity to make two sequential decisions. Smallville is relatively small, with fewer than 30,000 inhabitants. 

[STEPS]
1. Experimental design:
 We have employed two expected visibility conditions (low vs. high) in a between-subjects design.
2. Sample:
n = 50 shoppers (34 women) in a local supermarket in the city of “Smallville.” 
Participants were randomly assigned to two conditions, in a between-subjects design: high or low expected visibility of the consumed product. 
3. Measurements

(First decision: Purchasing a product that reflects social identity)
A sign displayed at the entrance to the supermarket invited shoppers to purchase a product (a short-sleeved shirt). In both cases, the city logo was printed on the shirt, reflecting participants’ social identity.

(Low-expected visibility condition)
The sign invited shoppers to buy a pajama top – reflecting low-expected visibility of the consumed product.

(High-expected visibility condition)
The sign invited shoppers to buy a t-shirt – reflecting high expected visibility of the consumed product.

(Second decision: Choosing a free gift)
A shopper who purchased a t-shirt was invited to choose a free pen. Two types of pens were offered: one expressing shoppers’ social identity (a pen printed with the city logo), and the other expressing their personal identity (a pen in a color of the shopper’s choice).
We coded a shopper’s second choice as “consistent” if he or she selected a pen printed with the city logo.
4. Hypotheses
H1: In a set of sequential identity-related product decisions, an individual is more likely to engage in consistent behavior—i.e., to make a second decision that emphasizes the same (personal or social) identity as the first decision—when the product involved in the first decision is expected to be consumed in high-visibility rather than low-visibility circumstances.
TITLE: DNA extraction from pinned specimens
AUTHORS: Caroline Storer, Demian F Gomez

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The purpose of this protocol is to extract DNA from pinned beetles.</div><div class = "text-block"><span>This protocol is part of the Bark Beetle Mycobiome (BBM) Research Coordination Network. For more information on the BBM international network: Hulcr J, Barnes I, De Beer ZW, Duong TA, Gazis R, Johnson AJ, Jusino MA, Kasson MT, Li Y, Lynch S, Mayers C, Musvuugwa T, Roets F, Seltmann KC, Six D, Vanderpool D, & Villari C. 2020. Bark beetle mycobiome: collaboratively defined research priorities on a widespread insect-fungus symbiosis. Symbiosis 81: 101–113 </span><a href="https://doi.org/10.1007/s13199-020-00686-9" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1007/s13199-020-00686-9</span></a><span>.</span></div></div>

[STEPS]
TITLE: Optima brain Mind Max Pills For Brain Boost 2022
AUTHORS: Brain  and Rain

[DESCRIPTION]
Optima brain Mind Max Pills For Brain Boost 2022


[STEPS]
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TITLE: CAMbank: SST Field Processing v1
AUTHORS: Eliah G Overbey, Krista A Ryon, JangKeun Kim, Christopher E Mason

[DESCRIPTION]
Field processing of SST vacutainers for the Cornell Aerospace Medicine Biobank (CAMbank).

Instructions for preserving: Serum and RBC Pellets.

[STEPS]
SECTION: Perform Venipuncture
1. After venipuncture, invert the tubes gently 8 to 10 times to fully mix tube anticoagulant with blood sample.

Store the tube upright at room temperature until centrifugation.

Allow the blood to clot in an upright position for at least 30 minutes. 
For optimal biochemical results, centrifuge and aliquot SSTs within 3 hours of venipuncture.
SECTION: Centrifuge Settings
2. Note: A swing bucket centrifuge is required. 

Set centrifuge:
acceleration: 9
deceleration: 9
temperature: RT
duration: 10 minutes
speed: 1300xg
SECTION: Centrifuge Settings
3. Place the SSTs in the centrifuge. 
Place a protective cover over the swing buckets in case of tube leakage. 
Start the centrifuge. 
Stand by the centrifuge until the centrifuge reaches max speed. Listen for signs of imbalance or compromised tube integrity. 
SECTION: Aliquot Serum
4. Carefully remove SSTs from the centrifuge and inspect for separation of red blood vs serum layers. 

Transfer SSTs to a sanitized laminar flow hood. 
SECTION: Aliquot Serum
5. Using a P1000, carefully aliquot serum into 2D barcoded tubes at 500µL each. 

Return 2D tubes with serum to the rack and place in the -80C freezer.
SECTION: Aliquot Serum
6. Recap and freeze the SST tubes to preserve DNA in the red blood cell pellet
TITLE: T cell depletion
AUTHORS: Connor Monahan

[DESCRIPTION]
This protocol details T cell depletion by antibodies targeting CD4 and CD8.

[STEPS]
SECTION: Procedure
1. Inject mouse intraperitoneally with 250 µg of Ultra-LEAF Purified anti-mouse CD4 or Rat IgG2b isotype control antibodies (Biolegend, Cat #100457 and Cat #400644; San Diego, CA) 3 and 1 days prior to immunization.
SECTION: Procedure
2. After immunization, inject mice with 250 µg of antibody weekly.
SECTION: Procedure
3. Assess cell depletion by flow cytometric analysis of mouse blood serum.
SECTION: Procedure
4. For depletion of CD8+ T cells, inject the mouse intraperitoneally with 250 µg of Ultra-LEAF Purified CD8+ or Rat IgG2b isotype control antibodies (Biolegend Cat # 100746 and Cat #400644; San Diego, CA) 2 days prior to immunization.
SECTION: Procedure
5. After immunization, inject mice with 250 µg of each antibody 9- and 19-days post immunization.

SECTION: Procedure
6. Assess cell depletion by flow cytometric analysis of mouse blood serum.
TITLE: Amplification of cytb gene of Plasmodium spp. in blood spots from wild animals
AUTHORS: Gabriela Ulloa Urizar

[DESCRIPTION]
Amplification of the Plasmodium mitochondrial cytochrome oxidase b gene (cytb) for detection of the Plasmodium genus in wildlife samples.

[BEFORE_START]
Materials
DW2 and DW4 primers
Platinum II Hot Start PCR Master Mix (2X)
Nuclease-free water
Template DNA (from blood spot samples)
PCR tubes
Thermal cycler
Agarose, TAE buffer
Gel electrophoresis system
DNA stain
DNA ladder (100 bp)


To detect Plasmodium spp. in humans and non human primates use the primers:
ROUND 1

DW2: 5'-TAA TGC CTA GAC GTA TTC CTG ATT ATC CAG-3'
DW4: 5'-TGT TTG CTT GGG AGC TGT AAT CAT AAT GTG-3'

Nested PCR cytb in humans and non human primates use the primers:
ROUND 2

FP3: 5'-TAT ATA ACT TAT TTT TTG ATA TG-3'
RP3: 5'-GTR ATW GCA TTA TCT GGA TGT GA-3'


For the PCR Mix (conventional and nested) we used the Platinum™ II Hot-Start PCR Master Mix (2X) from Invitrogen.


[GUIDELINES]
A) To detect Plasmodium spp. in humans and non human primates use the primers:

Procedure:
ROUND 1 PCR

1. Hydrate the primers with water without DNAases and dilute the primers for Plasmodium spp. (DW2 and DW4) to a concentration of 5uM.
 


2. Volumes for the pcr mix for 1 reaction (1 rx) were:

Primer DW2 [5uM]: 2 µL
Primer DW4 [5uM]: 2 µL
Master Mix (2X): 10 µL
Free water: 1 µL
Sample DNA: 5 µL

3. PCR conditions: 

94°C --- 3 min
94°C --- 20 s
62°C --- 30 s
72°C --- 1 min
The last 3 steps are repeated for 40 cycles. 
72°C --- 10 min
4°C --- store at this temperature until electrophoresis

Electrophoresis
4. Add 15 ul of PCR product to each well of the agarose gel. Then run on a 1.5% agarose gel prepared with TAE buffer at 95 volts for 40 min


 



B) nested PCR cytb in humans and non human primates use the primers:

ROUND 2 PCR

1.Hydrate the primers with water without DNAases and dilute the primers for Plasmodium spp. (FP3 and RP3) to a concentration of 5uM. 


 


2.Volumes for the pcr mix for 1 reaction (1 rx) were:

FP3 primer [5uM]: 2 µL
RP3 Primer [5uM]: 2 µL
Master Mix (2X): 10 µL
Free water: 4 µL
Diluted PCR product (1/20): 2 µL

3.PCR conditions:

94°C --- 3 min
94°C --- 20 s
52°C ---30 s
72°C --- 1 min
The last 3 steps are repeated for 40 cycles
72°C --- 10 min
4°C --- store at this temperature until electrophoresis


Electrophoresis
4. Add 15 ul of PCR product to each well of the agarose gel. Then run on a 2% agarose gel prepared with TAE buffer at 95 volts for45 min . Visualize bands under UV or blue light. Expected size: ~776 bp.



[STEPS]
TITLE: Miniprep Protocol (QIAGEN)
AUTHORS: Aditya Mohan

[STEPS]
?. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
?. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times.
?. Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA.
?. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times.
?. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. A compact white pellet will form.
Centrifuge: 17900 34
?. Apply 800 μl of the supernatant from step 4 to the QIAprep 2.0 spin column by pipetting.
?. Centrifuge for 30–60 s. Discard the flow-through.
?. Wash the QIAprep 2.0 spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through.
?. Wash QIAprep 2.0 spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.
?. Discard the flow-through, and centrifuge at full speed for an additional 1 min to remove residual wash buffer.
?. Place the QIAprep 2.0 column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB.
TITLE: Angiotensin-converting enzyme inhibitory assay
AUTHORS: Francisco Herrera

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The use of extracts obtained from various plants is an activity developed for years with the purpose of taking advantage of resources from the regions to prevent or cure a specific disease. On the other hand, the application of in vitro techniques, as angiotensin-converting enzyme inhibitory assay have allowed finding the relationship of organic compounds with biological activities and thus be able to determine which type of plants or their extracts are suitable to be used as a treatment for hypertension. In this protocol is described a method based on the colorimetric reaction of the hippuric acid with trichloro-triazine (TT).</div></div>

[STEPS]
?. Aqueous extract (40 µL) was added to a 20 µL solution of ACE (Sigma) (100 mU/mL), and incubated at 37 °C for 5 min.
?. Subsequently, 100 µL of HHL prepared at a concentration of 0.3% in a mixture of 40 µmol potassium phosphate buffer (JT Baker, Mexico)
?. Later 300 µM sodium chloride buffer (JT Baker, Center Valley, Pennsylvania, USA), previously adjusted to pH 8.3 either with 1 M HCl (JT Baker) or 1 M NaOH (JT Baker), and incubated at 37 °C for 45 min.
?. The reaction was stopped by adding 360 mL of TT dioxane (Sigma) solution (3% w/v) and 720 mL of 0.2 M phosphate buffer (JT Baker) (pH 8.3).
?. After centrifuging the reaction mixture at 10,000 × g for 10 min, enzymatic activity was determined in the supernatant by measuring absorbance at 382 nm (Perkin-Elmer, Lambda XLS, UV/VIS spectrophotometer).
?. The tests were performed in triplicate and antihypertensive activity was quantified by a regression analysis of ACE inhibitory activity (%) versus phenolic compounds concentration in aqueous extract and defined as an IC50 value, that is, the concentration of phenolic compounds required to produce 50% ACE inhibition under the described conditions.
?. For angiotensin-converting enzyme inhibitory assay lisinopril (antihypertensive drug) was used as control.
TITLE:     Direct ELISA for investigating the binding of  chemically-made Protein-LAG  to immunoglobulins.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protein LAG (SpLAG) is an immunoglobulin-binding protein that interacts with the Fc and Fab regions of many mammalian immunoglobulins. It is produced by a chemical coupling of individual proteins and them mixing it up to the appropriate protein ratio. SpLAG binds well to some avian immunoglobulin [1].</div><div class = "text-block">References</div><div class = "text-block">1.Vaillant AJ, McFarlane-Andersonv N, Wisdom B, Mohammed W, Vuma S, et al. (2013) Immunoglobulin-binding Bacterial Proteins (IBP) Conjugates and their Reactivity with Immunoglobulin in Enzyme-Linked Immunosorbent Assays (ELISA). J Anal Bioanal Tech 4: 175. doi:10.4172/2155-9872.1000175 </div></div>

[STEPS]
?. This ELISA is used to study the interaction of protein-LAG (SpLAG) with diverse immunoglobulins.
?. The 96 well microtitre plate is coated overnight at 4°C with 1 µg/µl per well of purified immunoglobulins or 50 µl of any animal sera in carbonate-bicarbonate buffer pH 9.6.
?. Then plate is treated with bovine serum albumin solution and washed 4X with PBS-Tween.
?. Then 50 µl of peroxidase-labeled-protein-LAG conjugate diluted 1:5000 in PBS-non-fat milk is added to each well and  incubated for 1.30h at RT. After that the plate is washed 4X with PBS-Tween.
?. Pipette 50 μl of 3,3',5,5' - tetramethylbenzidine (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 2 SD.
TITLE: MS2 Plaque Assay 
AUTHORS: Daniel  Ma

[DESCRIPTION]
MS2 plaque assay based on EPA Method 1601 and modified as a spot plating assay (Beck et al., 2009).

[STEPS]
SECTION: MS2 Plaque Assay
2. E. coli Famp Overnight Culture: Add 10 µL E. coli Famp (ATCC 700891) cyrostock to 10 mL  sterile Tryptic Soy Broth (30 g/L). Incubate . to obtain stationary phase.

SECTION: MS2 Plaque Assay
3. E. coli Famp Morning Culture: Add 0.75 mL E. coli Overnight Culture to fresh 100 mL  sterile Tryptic Soy Broth (3%) in a 250 or 500 mL flask. Incubate 150 min  . to exponential phase.
 


SECTION: MS2 Plaque Assay
4. Prepare Soft Tryptic Soy Agar: Prepare fresh agar on the morning of the plaque assay. Add 30 g/L Tryptic Soy Broth and 7.5 g/L Bacto Agar to distilled water. Mix vigorously. Sterilize in autoclave at 121 °C 45 min. Remove molten agar from autoclave and place in 48.5 °C water bath.
 


SECTION: MS2 Plaque Assay
1. Sterilize: Autoclave pipet tips and growth media at least two days before the plaque assay.
SECTION: MS2 Plaque Assay
5. Sample Preparation: Prepare MS2 samples by ten-fold serial dilution in micro-centrifuge tubes with 900 µL  1X PBS. Vortex samples between dilutions.

SECTION: MS2 Plaque Assay
6. Spot Plating Assay: After MS2 samples are diluted, prepare agar plates (10-15 mL) per plate to be used for spot plating.

First, prepare negative control plates containing only agar (no host cells).
Add 1 mL of host cells per 50 mL agar. Mix gently.
Add 10-15 mL of molten agar to bottom of Petri dish. Swirl to spread agar evenly across the bottom of the dish. Dry for 5-10 minutes or until the agar is solidified.
Label bottom of Petri dishes with sample information and draw grids for applying spots.
Spot MS2 samples onto the surface of the agar without touching the agar with the pipet tip. Apply the spots in labeled areas to keep track of sample information. Spot volumes can be anywhere from 1 µL to 50 µL. Incubate plates for 720 min to 960 min at 37 °C.
Remove plates from incubator and count plaque forming units (PFU) per spot for each sample and dilution. Record plaque forming units, dilution, sample identification, spot replicates, and spot volume. 
Calculate PFU/mL for each sample by aggregating total PFU across dilutions and accounting for the total undiluted volume: 

PFU/mL = (Total PFU across dilutions) / (Total Undiluted Volume of Sample across dilutions).
 
 

 

 



SECTION: MS2 Plaque Assay
6.1. This spreadsheet set up can be used for recording data and calculating concentrations.
TITLE: Neutralizing antibody detection
AUTHORS: Shihong Fu, Wenjing  Liu

[DESCRIPTION]
Neutralizing antibodies of serum samples were detected by 90% plaque reduction neutralization test (PRNT90).

[BEFORE_START]
the serum samples should be inactived (56℃，30min,in water)before the test.

[STEPS]
SECTION: Neutralizing antibody detection
1. 1. 	 Diluted serum: dilute the serum sample in a 96-well plate. Take 24 μl of inactivated serum and mix it with 96 μl of MEM culture medium to complete the 5-fold dilution of serum,the Serum samples were serially diluted two-fold beginning at a 1:5 dilution and ending at 1: 160 .
SECTION: Neutralizing antibody detection
2. 2. 	 Dilute virus: use MEM culture medium to dilute virus to 200 pfu/100 μl.
SECTION: Neutralizing antibody detection
3. 3. 	 The diluted serum (from 1:5-1:160) was mixed with an equal volume of 200 pfu of JE virus (strain P3). At the same time, different dilutions of virus (100 PFU, 10 PFU, 1 PFU) were set as controls, and the diluted virus solution was mixed with serum samples and incubated at 37 ° C and 5% CO2 for 1 h. 
SECTION: Neutralizing antibody detection
4.  4    Incubation of infected cells with the neutralized mixture: inoculate 6-well plates of BHK-21 cells grown to 90% confluence in monolayer, and incubate at 37℃, 5% CO2 for 1 h. 
SECTION: Neutralizing antibody detection
5. 5    Overlay methylcellulose medium culture: supplement 4 ml of 1.2% methylcellulose-MEM medium containing 2% FBS, culture at 37℃ and 5% CO2 for 3-5 days until obvious viral plaque is observed.
SECTION: Neutralizing antibody detection
6. 6. 	 Stain and calculate the neutralization: remove the culture medium, stain with crystal violet for 1h, gently rinse with running water, dry and count the number of viral plaques, and calculate the neutralizing antibody of JE virus in the sample.