eamag/protocols-with-steps huggingface dataset:
TITLE: Immunocytochemistry for the characterization of hiPSC to Motor Neuron differentiation
AUTHORS: Mallory Wright, ckremitz, William J Buchser

[DESCRIPTION]
This immunocytochemistry protocol is used for the characterization of IPSC differentiation into motor neurons using several biomarkers: neuroepithelial cells (SOX1), motor neuron progenitors (OLIG2 and NKX2.2), motor neurons (MNX1), and the mature motor neurons (ISL, ChAT, MAP2).

*Primary and secondary antibody information located in materials section
 





[STEPS]
1. Remove the medium from your cells 
 



2. FIX - Dilute 32% Paraformaldehyde solution to 4% PFA in 1X phosphate-buffered saline (PBS) 
3. Add 100uL of 4% PFA to each well in the 96-well plate. (1ml if using a 6-well plate). Incubate for 15 min at room temperature.

4. Remove the fixative solution and wash with 1XPBS at 100ul per well using a multichannel pipette. Repeat 3 times.
 

5. PERMEABILIZE - Add 100uL of 0.5% Triton X-100 to each well of a 96-well (1ml if using 6-well plate)
6. Incubate for 15 min at room temperature.

7. Remove the permeabilization solution and wash 3 times with 1XPBS 
8. BLOCK - Add 100ul of 3% BSA (bovine serum albumin) or blockAid-blocking solution to each well of a 96-well plate slowly to Block. (1ml if using 6-well plate)
9. Incubate for at least60 min (up to overnight) at room temperature.

10. Calculate the amount of primary antibody needed (located in materials section) and dilute in 3% BSA + 0.3% Triton X-100 solution 
11. PRIMARY ANTIBODIES - Remove 3% BSA from wells and add 100uL of primary antibody per well

 

12. Incubate for 60 min at room temperature in a dark place or overnight at 4°C.    
(Different primary antibody stains may take longer to stain and could require optimization).
13. Remove primary antibody and wash three times slowly with 1xPBS 
 

14. SECONDARY ANTIBODIES - Calculate amount of secondary antibody needed (located in materials section) and dilute in 3% BSA+ .3% TritonX-100 solution and 1:4000 Hoechst. 
15. Add 100uL per well in 96-well and incubate for at least 60 min at room temperature in a dark place.  

16. Remove secondary antibody and wash three times with 1xPBS 
17. Scan on the confocal microscope
TITLE: Making MES Buffers for Protein EDAC Particle Coupling 
AUTHORS: Kenneth Schackart, Kattika Kaarj

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to make MES buffers for covalently coupling proteins to carboxylated polystyrene particles via EDAC. Adapted from Bangs Laboratories Inc. TechNote 205 Covalent Coupling.</div><div class = "text-block"><span>Three buffers are made from 2-(</span><span style = "font-style:italic;">N</span><span>-morpholino)ethanesulfonic (MES) acid with varying pH. The three buffers are referred to as :</span></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Activation buffer (acidic for activating the carboxyl group on particles for EDAC reaction)</li><li style = "counter-reset:ol0;list-style-type:disc;">Coupling buffer (alkaline for coupling protein to o-acylisourea on particle surface created by EDAC reaction)</li><li style = "counter-reset:ol0;list-style-type:disc;">Storage buffer (for storing particles while preventing degradation and self aggregation)</li></ul></div></div>

[STEPS]
?. [Make MES Buffer Bulk]
Combine: MES free acid. DW.
12.8 g
600 mL
?. [Make MES Buffer Bulk]
Adjust to ~6 pH. If pH is low add NaOH, if pH is high add HCl.
Typically the solution is about   . In this case, adding about   of NaOH   should get you close. If the desired activation buffer pH is lower than   , adjust at this step to that pH.
?. [Separate and Adjust]
Separate the bulk buffer into the three buffers by transferring one third of the bulk to each of three glass bottles (~ each).
200 mL
?. [Separate and Adjust]
Adjust pH to the desired level for each buffer. NaOH is used to raise pH, HCl is used to lower pH. Recommended ranges from Tech Note 205 are shown below.Activation buffer:  -Coupling buffer:  -Storage buffer:
To reach ~  Coupling buffer, about      NaOH should be added.To reach ~  Storage buffer, about     NaOH should be added.
?. [Finish Storage Buffer]
Add bovine serum albumin (BSA) to make  to Storage buffer (not Activation or Coupling buffer).For  , add  BSA.Swirl to dissolve BSA.
200 mL
200 mg
BSA coats the particle surface, preventing nonspecific interaction (e.g. hydrophobic), also reducing self aggregation. The concentration of BSA can be varied from   -  . Additionally, surfactants may be used (e.g. Tween-80). Other blocking agents may be used in place of BSA.
?. [Store Buffers]
Store all buffers covered at  .
20 °C
TITLE: LessTests
AUTHORS: Ruth Polachek, doreen 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">LessTests SARS-CoV2 general diagnosis protocol, using LessTests software solution.</div><div class = "text-block">The LessTests method is agnostic to the labware, and has already been demonstrated as successful using multiple device and kit combinations. </div></div>

[STEPS]
?. Sample collection
Standard sample collection and transport using swabs
?. LessTests Encoder
Determines which samples are mixed into each pool.Optimizes pool number and other parameters to achieve best efficiency improvement.
?. Pooling using LessTests Method
Automated liquid handling workbenchPooling script provided by LessTests
?. Nucleotide extraction
RNA extraction kitNucleotide extraction system
?. Pool rRT-qPCR
rRT-PCR master mixqPCR reaction plates    SARS-CoV2 E, N, RdRP primersSARS-CoV2 E, N, RdRP probesReal time PCR system with software
?. LessTests decoder
Deciphers which sample are positive based on which pools were positive.
?. Inconclusive sample rRT-qPCR
When multiple samples can account for a positive results, they are flagged for an additional separate rRT-qPCR
?. Limit of detection
Determine maximal number of samples-per-pool. The results apply to the specific diagnostic setup tested, so needs to be done with the same equipment and kits used for later stages.2.1 Serial dilutions of samples with known Ct values.2.2 Nucleotide extraction2.3 rRT-qPCR
TITLE: Amyloid beta (Aβ) aggregates N-terminal labeling
AUTHORS: Patricia Yuste Checa, F Ulrich Hartl

[DESCRIPTION]
This protocol details how to efficiently label protein aggregates at the N-terminal using Amyloid beta as example. 

[STEPS]
SECTION: Amyloid beta (Aβ) aggregates N-terminal labeling
1. Centrifuge Aβ aggregates (e.g., 1 mL at 10 micromolar (µM)) at 20000 x g, 10 min, 4 °C.

SECTION: Amyloid beta (Aβ) aggregates N-terminal labeling
2.  Wash the pellet containing the aggregates with 1x PBS pH 7.2.

SECTION: Amyloid beta (Aβ) aggregates N-terminal labeling
3. Centrifuge at 20000 x g, 10 min, 4 °C.

SECTION: Amyloid beta (Aβ) aggregates N-terminal labeling
4. Wash the pellet with Labeling buffer (refer materials section).
SECTION: Amyloid beta (Aβ) aggregates N-terminal labeling
5. Centrifuge at 20000 x g, 10 min, 4 °C.

SECTION: Amyloid beta (Aβ) aggregates N-terminal labeling
6. Resuspend the pellet in 200 µL Labeling buffer.

SECTION: Amyloid beta (Aβ) aggregates N-terminal labeling
7. Dissolve the dye (Alexa488 (A488) NHS ester, Thermo Fisher Scientific, A20000; pHrodo Red Succinimidylester, Thermo Fisher Scientific, P36600) in DMSO.
SECTION: Amyloid beta (Aβ) aggregates N-terminal labeling
7.1. With a pipette tip gently touch the dye powder which will stick to the tip. 
SECTION: Amyloid beta (Aβ) aggregates N-terminal labeling
7.2.  Immerse the tip in some DMSO previously dispensed in a tube.
SECTION: Amyloid beta (Aβ) aggregates N-terminal labeling
7.3. Repeat the procedure several times until the solution reaches the desired color.
 

SECTION: Amyloid beta (Aβ) aggregates N-terminal labeling
8. Quantify diluted dye concentration by nanodrop. Dilute the sample in water to reach a λ<1 for an
accurate measurement.
 

SECTION: Amyloid beta (Aβ) aggregates N-terminal labeling
9. Add the corresponding amount of dye to a final protein:dye ratio of 1:10 (e.g., 50 µL of dye at 2.5 millimolar (mM) for 200 µL protein at 50 micromolar (µM)). 

SECTION: Amyloid beta (Aβ) aggregates N-terminal labeling
10. Incubate 60 min at Room temperature in the dark.

SECTION: Amyloid beta (Aβ) aggregates N-terminal labeling
11. Centrifuge at 20000 x g, 4 °C for 10 min.

SECTION: Amyloid beta (Aβ) aggregates N-terminal labeling
12. Wash the pellet with 1 mL methanol to remove excess dye (just when labeling with pHrodo dye).
 

SECTION: Amyloid beta (Aβ) aggregates N-terminal labeling
13. Centrifuge at 20000 x g, 4 °C for 10 min.
SECTION: Amyloid beta (Aβ) aggregates N-terminal labeling
14. Wash the pellet 2 to 4 times with 1x PBS pH 7.2.

SECTION: Amyloid beta (Aβ) aggregates N-terminal labeling
15. Resuspend the pellet with 1x PBS pH 7.2 or desired final buffer (e.g., in 200 µL buffer resulting in 50 micromolar (µM) labeled aggregates).

SECTION: Amyloid beta (Aβ) aggregates N-terminal labeling
16. Sonicate labeled aggregates in a Bioruptor sonication bath (Diagenode) (5 cycles of 5 seconds on – 5 seconds off), or similar.
SECTION: Amyloid beta (Aβ) aggregates N-terminal labeling
17. Aliquot, flash-freeze in liquid nitrogen and store at -80 °C.
TITLE: ELISA (enzyme-linked immunosorbent assay) for α-Syn quantification
AUTHORS: Jean-Louis Parmasad

[DESCRIPTION]
ELISA (enzyme-linked immunosorbent assay) for α-Syn quantification. Useful for analyses of alpha-Synuclein and related proteins in the context of synucleinopathy.

[STEPS]
1. Coat 384-well MaxiSorp plates (Nunc, Inc) with capturing antibody (α-Syn, BD Biosciences, 610787) diluted 1:500 in coating buffer (NaHCO3 with 0.2% NaN3, pH9.6) overnight at 4 °C.
2. Wash 3 times with PBS.0.05% Tween-20 (PBS-T). 
3. Block for 1hr at 37 °C in blocking buffer (1.125% fish skin gelatin; PBS-T)
4. Load samples in duplicate into wells.
5. Incubate at RT for 2hr.
6. Add the desired antibody to the plate for 1 h at 37 °C
7. Wash 5 times with PBS-T.
8. Add ExtrAvidin phosphatase (Sigma, E2636) diluted in blocking buffer to wells. Incubate for 30 min at 37 °C.
9. Carry out colour development with fast-p-nitrophenyl phosphate (Sigma, N1891) at 405 nm every 2.5 min for up to 60 min.
10. Identify time point(s) where standards and sample dilutions are in the log phase.
TITLE: Fluorescence microscope operation procedure
AUTHORS: 宏亮 董

[STEPS]
?. Production of temporary fitting
?. Add 15ul Bacterial solution to microslide at the clean workbench.
?. Add cover slide on it.
?. Use of fluorescence microscopy
?. Turn off the light in the room and turn on the microscope mercury lamp.
?. Select the appropriate filter according to the luciferin labeled with the sample.
?. Place the sample and find the right view. If you want to take a picture, please make sure that the camera is loaded with color film.
?. Turn on the selfie device and select the manual file, usually within 0.5 to 10 seconds.
?. End of use, turn off all power and record use.
TITLE: Human metagenomics protocols Payami lab
AUTHORS: Haydeh Payami

[DESCRIPTION]
This is a collection of protocols that details the entire process of conducting a human microbiome study from start to end. It includes (1) consent form, to enroll subjects with permission to store and share data (2) data collection protocols designed to be self-administered by subjects (Environmental & Family History Questionnaire, Gut Microbiome Questionnaire, instructions for saliva collection, and instructions for stool collection), (3) codes for bioinformatics and statistical data analysis, (4) workflow at a glance created according to STORM guidelines and (5) final product, the manuscript, which describes the whole process from start to end using metagenomics of Parkinson’s disease implicates the gut microbiome in multiple disease mechanisms.

[STEPS]
TITLE: SaCas9 protein purification
AUTHORS: Fatma Gomaa, Zhu-Hong Li, Roberto Docampo, Virginia Edgcomb

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify"><span>Developing transfection protocol for </span><span style = "font-style:italic;">Bodo saltans</span><span>, using SaCas9/sgRNA ribonucleoprotein (RNP) complex in conjunction with DNA repair template to disrupt the Paraflagellar rod 2 gene (</span><span style = "font-style:italic;">BsPFR2</span><span>) and increase the efficiency of targeted homologous recombination when a repair template DNA is provided. The exogenous repair template is double stranded DNA and it consists of </span><span style = "font-style:italic;">eGFP</span><span> fused with the drug selection gene </span><span style = "font-style:italic;">nptII/neo </span><span>and flanked by 500 bp of the untranslated regions (UTRs) upstream and downstream of the targeted </span><span style = "font-style:italic;">BsPFR2</span><span> as homologous repair arms.</span></div><style>
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[STEPS]
?. [SaCas9 protein purification]
Protocol 1: for SaCas9 protein purification SaCas9 protein was prepared following methods described in Medeiros et al. (Medeiros et al., 2017) with modifications. Briefly, the bacterial expression vector p6XHis_NLS-SaCas9 (Addgene #101086) was transformed into Escherichia coli Rosetta 2(DE3) competent cells (Novagen) and grown as an overnight preculture with shaking at 37°C. Subsequently, 1 ml of preculture was used to inoculate 100 ml of terrific broth media which was incubated with shaking at 37°C until it reached an optical density at of 0.6 at 600 nm. Protein expression was induced by addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 200 µM and cells were grown overnight with vigorous shaking at 18°C. Following incubation, cells were collected by centrifugation, and the pellet was resuspended in 2 ml / 100 mg cell weight of xTractor Buffer (Takara Bio) plus 5 mg of lysozyme. Cells were lysed by sonication, then 1 µl / 2ml of DNAse I was added. The soluble fraction was purified using the His60 Ni Superflow Resin & Gravity Columns (Takara Bio) following the manufacturer’s recommendations. The concentration of SaCas9 containing fractions were determined by optical density at 280 nm and then pooled. Pooled fractions were concentrated and desalted into Cytomix (120 mM KCl, 0.15 mM CaCl2, 10mM KH2PO4, 25 mM HEPES, 2 mM EGTA, 5 mM MgCl2, pH 7.6) using Amicon Ultra-15 centrifugal filter devices (Millipore Sigma). The protein concentration was determined using a BCA Protein Assay Kit (Pierce).
TITLE: OpenVent DNA Polymerase Production
AUTHORS: Jenny Molloy, Stephane Fadanka, Nadine             Mowoh

[DESCRIPTION]
This collection contains the protocols for production, quality control, packaging and use of Beneficial Bio's dehydrated OpenVent DNA Polymerase.

Format: 32 tubes (4x8-tube PCR strips) 20ul of Enzyme each

OpenVent DNA polymerase is an extremely high thermostable and high fidelity enzyme suitable for routine PCR applications and amplification of GC-rich or looped sequences. The enzyme is compatible with a wide range of templates and its robustness guarantees reliable amplification results in almost all PCR applications. The Eco and room temperature stable format - PCR reaction tube strips containing the dehydrated enzyme (10 reactions per tube), offers affordability and flexibility.

FEATURES
Dehydrated formulation enables better stability at room temperature
Ideal for a wide range of applications including GC-rich or looped sequences amplification
Amplification of low copy DNA targets
Eco formulation and flexible format

DOCUMENTS

Product Manual
Product Specification Sheet (PSS)
Certificate of Analysis (CoA)

 
 

[STEPS]
TITLE: Bacterial Culture for Plasmid Extraction
AUTHORS: Hung Liang Pai, Huan Jui Chang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">In this protocol, we present the method of culturing bacteria overnight after transformation. This protocol will typically undergo plasmid extraction and baterial storage in the ensuing steps. </div></div>

[STEPS]
?. [Preparation]
Turn on the UV light of the laminar flow for  to   in order to sterilize it.
?. [Preparation]
Prepare LB Media.
?. [Preparation]
Add 50 mL of LB Media to a 50 mL of centrifuge tube.
?. [Preparation]
Add 50 μL of chloramphenicol.
?. [Protocol]
Use a toothpick to pick a single colony from a LB agar plate. Make sure the tip of the toothpick contains bacteria.
?. [Protocol]
Add 7 mL of LB Media containing choramphenical to a 15 mL centrifuge tube
?. [Preparation]
Gently shake the tube to mix.
?. [Preparation]
Prepare a LB Agar Plate with transformed bacterial colonies on it.
?. [Protocol]
Drop the toothpick into the 15 mL centrifuge tube. Make sure the tip of toothpick fully submerge in the LB Media.
?. [Protocol]
Incubate the 15 mL centrifuge tube for  .
[16-18 hour]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
For <a href="https://www.protocols.io/view/Enumeration-of-virus-particles-in-aquatic-or-sedim-dpy5pv" target="_blank">Enumeration of virus particles in aquatic or sediment samples by epifluorescence microscopy</a> protocol.

[GUIDELINES]
<strong>Preservation of samples (SYBR only):</strong> If slides cannot be made in the field on freshly collected samples, preservation will be necessary. Many earlier estimates of viral abundance have been made on preserved samples; however, several studies have shown rapid and significant decay of viral particles in aldehyde-fixed samples (Brussaard 2004; Danovaro et al. 2001; Wen et al. 2004), although there is evidence that the decay is lessened when larger-volume samples are preserved (Patel et al. 2007). Samples preserved in the field should be flash-frozen in liquid nitrogen. Even freezing with dry ice (frozen CO<sub>2</sub>) or immediately freezing in a –87°C freezer is not adequate for preservation, and to our knowledge, freezing in a dry ice/ethanol slurry (–78°C) has not been tested. Samples to be preserved should be fixed in 0.5% (final concentration) EM-grade glutaraldehyde and flash-frozen in liquid N<sub>2</sub>. For relatively productive samples with virus abundances ≥10<sup>6</sup> mL<sup>–1</sup>, add 30 µL of 25% EM-grade glutaraldehyde into prelabeled 2-mL cryovials, add 1470 µL of the sample to be counted, and mix. These volumes can be halved for samples with virus abundances ≥10<sup>7</sup> mL<sup>–1</sup> and increased to 4 mL in 5-mL cryovials for very oligotrophic samples. Allow the samples to stand for at least 15 min but no longer than 30 min, and then freeze immediately in liquid N<sub>2</sub>. To facilitate easy retrieval, vials can be placed in women’s nylon stockings before freezing. (We find black sheer ones the most aesthetically pleasing.) Once frozen, the vials can be transferred to –80°C for long-term storage, until the slides can be prepared. For counting, the frozen samples are thawed in a 37°C water bath and immediately stained with SYBR Green I or SYBR Gold, as outlined.<br /><br />In the field, if it is impossible to make slides or preserve in liquid N, then it probably is best to collect the samples in as large volumes as is practical and maintain them under in situ conditions for as short a time as is possible until slides can be made.

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
We use this process to create our own Luminex assays from standard ELISA reagents.

[BEFORE_START]
Activation buffer (100 mM NaH2PO4, pH 6.3)<br />Coupling buffer (50 mM HEPES, pH 7.4)<br />PBS<br />PBS/1% BSA<br />EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide)<br />S-NHS (N-hydroxysulfosuccinimide)

[GUIDELINES]
Minimize the exposure of EDC and S-NHS to air and moisture. Use fresh aliquots for each coupling reaction and discard after use. Pierce sells individually packaged, small amounts of S-NHS that can be used in a single use fashion.

[STEPS]
?. 
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Cognitive theories of depression posit that negative schemata constrain how emotional information is attended to, processed and recollected  (Beck et al., 1979).    Numerous studies have demonstrated an association between acute depression and preferential processing of negative information and/or impaired processing of positive emotional information (Bradley et al, 1995; Gilboa-Schechtman et al., 2002; Gur et al., 1992; Peckham et al., 2010; Ridout et al., 2003; Surguladze et al., 2004) which persists into periods of remission (Anderson et al., 2011; Bhagwagar et al,  2004; Hayward, et al., 2005).  Emerging evidence also suggests that these behavioural abnormalities are present prior to the onset of illness and therefore reflect a trait vulnerability marker for depression.  For example, Chan and colleagues (Chan, et al., 2007) reported that high neuroticism (a recognised risk factor for depression) was associated with reduced latency to classify negative vs. positive personality descriptors, reduced positive memory intrusion at subsequent recall and reduced capacity to recognise happy facial expressions as compared to individuals with low levels of neuroticism.  We have also shown that late chronotype, also recognised as a risk factor for depression, is associated with increased capacity to recognise sad vs happy facial expressions (Berdynaj et al., 2016; Horne, Marr-Phillips, Jawaid, Gibson, &amp; Norbury, 2017).<br />             Less well understood is whether bias towards emotionally positive stimuli relates to resilience. The concept of resilience can be thought of as a stress coping ability with  positive adaptation and outcomes in the face of adversities (Connor &amp; Davidson, 2003) and should act as a buffer against the harmful effects of future stressors. In support of this notion Sherrin and colleagues (Sheerin et al., 2017) in a  longitudinal study reported that higher resilience was a protective factor against future internalising psychopathology. Moreover, using the dot-probe task to assess individual differences in attentional bias towards happy or angry facial expressions Thoern et al., (Thoern, Grueschow, Ehlert, Ruff, &amp; Kleim, 2016) observed that healthy participants with a greater attentional bias towards happy faces reported higher trait resilience. This finding may have implications for a number of stress-related disorders and suggests that increasing the tendency to attend to emotionally positive stimuli could render individuals more resilient. However, Thoern and colleagues did not take into account individual differences in neuroticism, circadian preference or depressive symptomatology (three recognised risk factors for depression). The aims of the current proposal, therefore, are to: 1) replicate the findings of Thoern and colleagues, i.e. that increased attention to positive emotional stimuli predicts trait resilience, 2) extend this work to include emotion recognition, and 3) explore the interaction between risk factors for depression, resilience and attentional bias/emotion recognition </p>

[STEPS]
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