eamag/protocols-with-steps huggingface dataset:
TITLE: High GC-content PCR
AUTHORS: Joao Vitor Molino

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">X7 is a a Pfu-sso7d fusion DNA polymerase that is compatible with uracil-excision cloning and has been used with sucess to amplify our high GC-content sequences and it is a valuable tool for USER clonning.</div><div class = "text-block">Before starting</div><div class = "text-block"><span>Please note that protocols with X7 may differ from protocols with other polymerases. Conditions recommended below were tested with our sequences, mostly high GC-content sequences from </span><span style = "font-style:italic;">Chlamydomonas reinhardtii </span><span>expression vectors. </span></div><div class = "text-block">Please refer to the original work that develop the fusion polymerase. </div><div class = "text-block">All components should be mixed prior to use. </div><div class = "text-block">Reference:</div><div class = "text-block"><span>Nørholm, M. H. H. (2010). A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering. BMC Biotechnology, 10. </span><a href="https://doi.org/10.1186/1472-6750-10-21" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1186/1472-6750-10-21</span></a><span style = ":;"></span></div></div>

[STEPS]
?. [Reaction preparation]
Set up the reaction using the following table on ice:   AB1ReagentVolume (uL)2Phusion® GC Buffer (NEB) 5X103Betaine 5M104dNTPs 10 uM1510 µM Forward Primer2.5610 µM Forward Primer2.57Template DNAVariable (~0.5-1)8X70.59Nuclease-Free Water2310Final Volume50Mix the reaction gently and spin it down if necessary.
AB1ReagentVolume (uL)2Phusion® GC Buffer (NEB) 5X103Betaine 5M104dNTPs 10 uM1510 µM Forward Primer2.5610 µM Forward Primer2.57Template DNAVariable (~0.5-1)8X70.59Nuclease-Free Water2310Final Volume50
?. [Reaction preparation]
Transfer PCR tubes to a thermocycler and start the PCR program. Below it is presented a default setup which can varied accordingly to the target amplicon. The recommended cycle setup and temperatures are shown below.   ABC1STEPTEMPTIME2Initial Denaturation98 °C30 seconds335-40 Cycles98 °C10 seconds460 °C20 seconds568 °C1 minute/kb6Final Extension72 °C5 minutes7Hold4 – 10 °C
ABC1STEPTEMPTIME2Initial Denaturation98 °C30 seconds335-40 Cycles98 °C10 seconds460 °C20 seconds568 °C1 minute/kb6Final Extension72 °C5 minutes7Hold4 – 10 °C
TITLE: Immunological detection of autophagy and mTORC1-related proteins
AUTHORS: Harper    JW, Sharan Sharan Swarup

[DESCRIPTION]
Here we present a general protocol for immunological detection by Western blotting MTOR, MTOR (pS2448), ULK1, ULK1 (pS757), p70S6K, p70S6K (pT389), SQSTM1, CALCOCO2, MAP1LC3B, GABARAP, TFEB, TFE3, PGRN, HSP90, and PCNA

[STEPS]
SECTION: Western blotting
1. Lyse cell pellets by homogenization in KPBS buffer, urea buffer, or RIPA buffer with protease and phosphatase inhibitors. FOr some experiments, we employ 293 cells or alternatively HeLa cells with or without the GRN gene, created by CRISPR-based gene editing (DOI: dx.doi.org/10.17504/protocols.io.4r3l2oxqqv1y/v1). 
SECTION: Western blotting
2. Determined total protein concentration by BCA or Bradford assay.  Normalize samples within a set of samples with additional buffer.  Add NuPAGE LDS buffer (4X) plus NuPAGE reducing agent (10X).
SECTION: Western blotting
3. Load samples onto 4-12% NuPAGE Bis-Tris gels (ThermoFisher), and separate by electrophoresis in MES buffer.
SECTION: Western blotting
4. Transfer proteins to PVDF or nitrocellulose membranes by standard wet transfer in 20% methanol.
SECTION: Western blotting
5. Stain membranes with REVERT 700 total protein stain following manufacturer’s instructions, and image total protein with a ChemiDoc MP (Bio-Rad) at 680 nm.
SECTION: Western blotting
6. De-stain with REVERT reversal solution for 5 min. Block membranes with tris-buffered saline (TBS) (5% non-fat dry milk) at room temperature for 30-60 min.
SECTION: Western blotting
7. Incubate membranes overnight at 4ºC with primary antibody solution in TBS with 0.1% Tween-20 (TBST). Wash six times with TBST for 5 min each.  Incubate in secondary antibody solution in TBST (plus 0.01% SDS) for 1h at room temperature.
SECTION: Western blotting
8. Wash membranes four times with TBST for 5 min each.
SECTION: Western blotting
9. When using HRP-conjugated secondary antibodies (Bio-Rad or Cell Signaling Technology), apply luminol and hydrogen peroxide solution to membrane for 2 min, and image membrane with a ChemiDoc MP using the chemiluminescent setting.
SECTION: Western blotting
10. When using Li-Cor fluorescent secondary antibodies, blot membranes dry and image with a ChemiDoc MP at either 800 nm or 680 nm, depending on the secondary antibody.
TITLE: Genetic expression suppressor screen
AUTHORS: Alexandros C Kokotos, Tim Ryan

[DESCRIPTION]
This protocol describes a low-throughput genetic screen to identify glycolytic enzymes that improve neuronal function under hypometabolic conditions.

[STEPS]
SECTION: Neuronal culture and transfection
1. Use cultured rodent primary hippocampal neurons, transfected with vGlutI-pH, mTagBFP2 and a construct expressing a single glycolytic enzyme, as explained in dx.doi.org/10.17504/protocols.io.ewov1qxr2gr2/v1.

SECTION: Live-cell imaging
3. Mount the coverslips with the cells on the imaging chamber and image as explained in dx.doi.org/10.17504/protocols.io.q26g7pn4qgwz/v1.
SECTION: Live-cell imaging
2. Remove the cells from the incubator and bathe them in Tyrodes containing 0.1 mM glucose.
SECTION: Live-cell imaging
4. Locate cells in an unbiased manner using the mTagBPF2 channel and center on their axon, judging by their morphology.
SECTION: Live-cell imaging
5. After a total of 5 min that the cells have been in the 0.1 mM glucose tyrode buffer, start the live-cell imaging experiment in the vGlutI-pH channel.

SECTION: Live-cell imaging
6. Stimulate the cells with 100 APs delivered at 10 Hz every 1 min, for a total of 10 rounds of activity.

SECTION: Live-cell imaging
7. At the end of the experiment, perfuse cells with Tyrodes containing 50 mM NH4Cl.
SECTION: Live-cell imaging
8. Analyze the collected data accordingly.
TITLE: Ultracentrifuge clean-up of phage lysate for TEM
AUTHORS: Kathy Lam

[STEPS]
?. Take 1.1 ml of existing plate lysate
?. Take 1 ml to clean up; save remainder to titer pre- versus post-clean up
?. Transfer 1 ml to ultracentrifuge tube
?. Centrifuge 25,000 rpm (~25,000g) 60 min 4C
?. Pour off supernatant away from pellet; pipette off residual
?. Wash with 0.1 M ammonium acetate
?. Centrifuge 25,000 rpm (~25,000g) 60 min 4C
?. Pour off supernatant away from pellet; pipette off residual
?. Wash with 0.1 M ammonium acetate
?. Centrifuge 25,000 rpm (~25,000g) 60 min 4C
?. Pour off supernatant away from pellet; pipette off residual
?. Resuspend in 0.1 M ammonium acetate
?. Titer pre- and post-clean up samples
?. Store at 4C for up to 2 weeks
TITLE: PI staining of Arabidopsis seedling
AUTHORS: Magdalena Julkowska

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

[STEPS]
?. [transfer the seedlings from agar plate into a solution containing the nutrients as well as 5-10 µg/mL of propidium iodide. Incubate for 20 to 30 min.]
?. [Examine the red staining under (confocal) microscope]
?. [Examine the red staining under (confocal) microscope]
TITLE: In vitro GCase activity assay (total cell lysate)
AUTHORS: Federico   Bertoli, Michela Deleidi

[DESCRIPTION]
Glucocerebrosidase is a lysosomal enzyme that catalyzes the hydrolysis of glucosylceramide (GlcCer), a membrane glyco-sphingolipid, to ceramide and glucose. This assay detects GBA activity by using a fluorogenic substrate that reacts with cell lysates previously treated with or without CBE (GBA1 inhibitor).

[STEPS]
SECTION: Sample Lysis
1. Suspend samples in 50 µL of 1% Triton extraction buffer.

SECTION: Sample Lysis
2. Homogenize with a Dounce homogenizer for 25 strokes.
SECTION: Sample Lysis
3. Rotate samples for 30 min at 4 °C.

SECTION: Sample Lysis
4. Centrifuge at 13500 x g,  4 °C for 15 min.

SECTION: Sample Lysis
5. Collect supernatants.
SECTION: Substrate preparation
6. Add 20.30 mg 4-Methylumbelliferyl-β-D-glucopyranoside for 10 mL ddH2O of substrate (6 millimolar (mM)).

SECTION: Substrate preparation
7. Incubate at 55 °C and vortex every 5 min until dissolved (approx. 30 min).

SECTION: Substrate preparation
8. Store at 4 °C until needed.

SECTION: Sample preparation
9. 
Add the equivalent of 10 µg total protein in ddH2O to reach a final 45 µL volume.
 


SECTION: Sample preparation
10. Add to each 25 µL McIlave Buffer pH 6 and mix it. 


 

SECTION: Sample preparation
11. Divide the overall 70 µL volume into two tubes (35 µL each).

SECTION: Sample preparation
11.1.  Incubate one tube with 5 µL  CBE  1 millimolar (mM) at  4 Room temperature  for   30 min .

SECTION: Sample preparation
11.2. Incubate the other one with 5 µL ddH2O at 4 Room temperature for 30 min.

SECTION: Enzymatic reaction
12.  Add 25 µL substrate to each reaction tube.

SECTION: Enzymatic reaction
13. Incubate at 37 °C for 120 min.

SECTION: Measurement
14. Take 10 µL  of each reaction tube into a 96-well plate (in triplicate).

SECTION: Measurement
15. Add 90 µL  0.2 Molarity (M)  glycine pH 10.2 to each well to stop the reaction.

SECTION: Measurement
16. Measure fluorescence: Excitation 355nm, Emission 460nm.
TITLE: Mobility, well-being, and community participation among people with depression: an observation study using geographically-explicit ecological momentary assessment (GEMA)
AUTHORS: Maritta A Välimäki, Man Sing Wong, Thomas Choi, Paul Lee, Lin Yang, Rick Kwan, Oscar Chung, Xinyu Yu, Rui Zhu, Sau-fong Leung

[DESCRIPTION]
Information on the mobility and daily routines of persons with depression remains limited. Our goal was to describe a preliminary explication of the relationship between mobility, well-being, and community participation of people with depression, and the relationship between mobility and environmental measures on well-being. We used a multivariate, time-series, single-subject, repeated measures design. People with depression (N=33) used a GPS device two weeks for mobility tracking, and the records were extracted from Geographical Information Systems (GIS). Logistic regression of mobility and environmental measures on well-being outcomes were adjusted for age and sex. The correlation coefficients showed that the more hours participants (N=32) stayed at home, the more often they had depressive symptoms, their satisfaction with life was still higher. Participants with longer total distance reported more stress. The fewer destinations participants travelled to, the more depressive symptoms they had. Time spent outside home was also negatively associated with satisfaction with life and self-esteem. Our work showed that depressive symptoms may limit people’s willingness to move outside home although the pattern is not clear. More studies should be conducted to verify our assumption with a larger sample size. 

[STEPS]
TITLE: Patch-Seq Recording and Extraction
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the process to obtain electrophysiological recordings and cellular contents from neurons in postnatal mouse and/or human brain slices.</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: Phonological Awareness tasks for Italian-English comparison
AUTHORS: Valeria Marinelli, Pierluigi Zoccolotti, Cristina Romani

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:center"><span style = "font-weight:bold;">Phonological Awareness tasks</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:center"></div></div><div class = "text-block">An attempt was made to develop tasks that would be comparable between the Italian (i.e., a consistent orthography) and English (i.e., an inconsistent orthography)versions. </div><div class = "text-block"><div class = "justify" style = "text-align:justify"><span style = "font-style:italic;">Phonological awareness </span><span>was measured using a spoonerisms and a phoneme deletion task. Typically, there is a relatively narrow range of performance on phonological awareness tasks in readers of regular languages (</span><span style = "font-style:italic;">e.g.</span><span>, Cossu et al., 1988) and this may limit their predictive power. For this reason, we used two tests (phoneme deletion and spoonerisms tasks) not susceptible to ceiling effect and sensitive to capture individual differences in older children and adults (Bruck, 1992; de Jong & van der Leij, 2003; Hatcher, Snowling & Griffiths, 2002; Hulme et al., 2002; Landerl et al., 1997; Paulesu et al., 2001; Ramus et al., 2003), as well as in very young children (Caravolas et al., 2005). </span></div><style>
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[STEPS]
TITLE: Growth curves
AUTHORS: Elizabeth Fozo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Rules of thumb and directions for making growth curves</div></div>

[STEPS]
?. [Rules of thumb]
Never remove > 1/3 of a culture for a growth curve
?. [Rules of thumb]
Read OD600 (optical density at 600 nm) of 200 microliters of culture added to 800 microliters of fresh media → 1,000 microliters of fluid in cuvette; = 1:5 dilution
?. [Rules of thumb]
Easier to measure 800 microliters of fresh media into cuvettes ahead of time; simply store in cuvette box
?. [Rules of thumb]
Easier to measure media into a 50 mL conical for filling fresh cuvettes; less likely to get a contaminated bottle
?. [Rules of thumb]
Easier to use same blank multiple times; suggest every time you refill conical with media for cuvettes, make a new blank with that and use with cuvettes from that mixture.
?. [Rules of thumb]
Place cuvettes into 10% bleach solution when finished *This is a must for BSL 2 organisms such as E. faecalis.
?. [Making an overnight culture]
Measure 5mL appropriate media with appropriate antibiotics into fresh conical (make sure to label conical with date, strain!)
?. [Making an overnight culture]
Take 1 colony from a plate with appropriate strain (use pipette to poke that colony)
?. [Making an overnight culture]
Inoculate media
?. [Making an overnight culture]
Grow overnight (usually start culture ~3-5 p.m. night before culture is needed)
?. [In the morning:]
Measure OD600
?. [In the morning:]
Use V1C1=V2C2 to make a 0.01 OD600 dilution in media for growth curve
?. [In the morning:]
Place culture in flasks, mix, place in appropriate incubator, start count up timer.
?. [Growth curve:]
The first time point is at 30 min on count up timer.
?. [Growth curve:]
Take time point every 30 min, write down data and graph as you go
?. [Growth curve:]
Use the count-up timer to measure time. When it reaches 30 min, 1 hour, 1 ½ hours, etc. remove cultures from incubator, pipette into cuvettes (remember 1/5 dilution, e.g. 200 microliters culture in 800 microliters media)
?. [Growth curve:]
Remember to mix culture gently before adding to cuvette (the culture can be clumpy at the bottom, and you want a homogenous solution)
TITLE: Left atrial function by speckle tracking echocardography in HFpEF
AUTHORS: RC Rimbas, SL Magda, S Mihaila-Baldea, ML Luchian, AM Chitroceanu, M Hayat, DJ Mihalcea, R Dragoi-Galrinho-Antunes-Guerra, M Stefan, A Velcea, A Andronic, L Lungeanu-Juravle, AI Nicula, D Vinereanu

[DESCRIPTION]
AIMS. None of the conventional echocardiographic parameters alone predict increased NTproBNP level and symptoms, making diagnosis of heart failure with preserved ejection fraction (HFpEF) very difficult in some cases, in resting condition.
We will evaluate LA functions by 2D speckle tracking echocardiography (STE) on top of conventional parameters in HFpEF and preHF patients with diastolic dysfunction (DD), in order to establish the added value of the LA deformation parameters in the diagnosis of HFpEF.
METHODS. We will enroll  patients with HFpEF  and compare them with asymptomatic patients with similar risk factors with DD (preHF). We will evaluate them by NTproBNP, conventional DD parameters, and STE. Global longitudinal strain (GS) was added. LA reservoir (R), conduit (C), and pump function (CT) were assessed both by volumetric and STE. 2 reservoir strain (S) derived indices were also measured, stiffness (SI) and distensibility index (DI). 

[STEPS]
SECTION: LA function in FpEF
1.
TITLE: DNA extraction from sputum
AUTHORS: Laszlo Irinyi, Lana Pasic, Wieland Meyer

[STEPS]
?. [Breaking the cells with liquid nitrogen]
The sputum samples were transferred to 1.5ml Eppendorf tubes for liquid nitrogen cell wall breakage, with a maximum of 600 µl of sputum per tube. Under full PPE conditions, the lid of the tube was left open, and a small plastic pestle placed to sit inside. Liquid nitrogen was transferred into a 100 ml cup. The tube, with the pestle inside, was placed into the liquid nitrogen, up until the lid opening, for three seconds. Once removed, the pestle was used to ground up and crush the sputum inside, turning it continuously for 60 seconds, and the process was repeated five times for each tube.
?. Add 20 L of both Proteinase K and Lysozyme (user supplied) to the precipitated slurry pellet resulting from the sputum sample. Vortex for 10 seconds.
?. [Following the manufacturer’s instructions of Sputum DNA Isolation Kit (Cat. 46200, Norgen BioTek Corp., Thorold, ON, Canada)]
Add 4 mL of Slurry D for every 1 mL of sputum sample in a 15 mL conical tube. Mix well by inversion. (Note: Slurry D contains silicon carbide resin and must be mixed well before every pipeting).
?. Centrifuge for 5 minutes at 2,000 RPM. Discard the supernatant.
?. Incubate the mixture at 60C for 20 minutes.
?. After the 20 minute incubation, add 260 L Solution WN, and mix well by pipetting up and down.
?. Transfer the entire contents into a Mini Filter Spin column (provided) with collection tube.
?. Centrifuge for 1 minute at 14,000 RPM, and discard the flow-through.
?. Apply 500 μL of Wash Solution BE to the column and centrifuge for 1 minute. Discard the flowthrough and reassemble the spin column with its collection tube.
?. Apply 500 μL of Wash Solution BE to the column and centrifuge for 1 minute. Discard the flow-through and reassemble the spin column with its collection tube.
?. Apply 500 μL of 96-100% Ethanol (user supplied) to the column and centrifuge for 1 minute. Discard the flow-through and reassemble the spin column with its collection tube.
?. Spin the column for 2 minutes in order to thoroughly dry the resin. Discard the collection tube.
?. Transfer the spin column to a fresh 1.7 mL Elution tube. Apply 100 L of Nuclease free water to the column and centrifuge for 2 minutes at 2,000 RPM, followed by 1 minute at 14,000 RPM.
?. The DNA concentration of the samples is determined using DeNovix dsDNA Broad Range Fluorescent Assay Kit (Denovix, Wilmington, USA)
TITLE: Immunoprecipitation (IP)
AUTHORS: nguyen.tha

[DESCRIPTION]
This protocol details about immunoprecipitation using anti-HA magnetic beads.

[STEPS]
SECTION: Procedures
1. Lyse cell pellets (5-7 mg) in 500 µL IP lysis buffer containing IP base buffer supplemented with 1x cOmplete, EDTA-free protease inhibitor cocktail and 0.1 µL of benzonase and incubate samples on ice for 30 min. Mix the sample by inverting the eppies gently every 5 min.
SECTION: Procedures
2. Wash anti-HA beads with 500 µL of bead equilibration buffer.

SECTION: Procedures
4. Centrifuge the cell lysates at max speed for 10 min at 4 °C.

SECTION: Procedures
5. Carefully transfer cleared lysates into 2 ml eppies and take 50 µL from each tube for “Input” samples.

SECTION: Procedures
6. Gently add 1000 µL of IP base buffer containing 1x cOmplete, EDTA-free protease inhibitor cocktail to the rest of each sample to dilute out the detergent.

SECTION: Procedures
7. Incubated the diluted cleared lysates with the anti-HA magenetic beads on a rotary mixer for 180 min at 4 °C.

SECTION: Procedures
8. Collect beads on a magnetic rack and aspirate the unbounds.
SECTION: Procedures
9. Wash with 1 mL IP wash buffer.

SECTION: Procedures
10. Repeat steps 7-8 another 4 times.
 

SECTION: Procedures
11. Elute with 25 µL elution buffer by boiling at shaking at 99 °C for 10 min.
SECTION: Procedures
3. Repeat step 2 twice.
TITLE: Quality Control and Data Recording for DDNS
AUTHORS: Alex Shaw, Joyce Akello, Catherine Troman, Aine.OToole, Erika Bujaki, c.ansley, Zoe Vance, rachel.colquhoun, Andrew Rambaut, Javier Martin, Nick Grassly

[DESCRIPTION]
This standard operating procedure indicates how to perform quality control checks for DDNS and provides guidance on best practice for ensuring relevant sequencing run data are recorded. The document is structured into quality control steps for setting up data files for DDNS, the RT-PCR and library preparation, and the sequencing run.




[STEPS]