eamag/protocols-with-steps huggingface dataset:
TITLE: Making glycerol stocks of bacteria
AUTHORS: Cristian Riccio

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Freezing down a bacterial culture for long-term storage.</div></div>

[STEPS]
?. Make sure you have autoclaved glycerol, cryovials and a fresh bacterial culture in liquid b-broth or LB. If you don't have a fresh bacterial culture, you can grow them in b-broth or LB medium at 180 rpm in flasks.
?. Prepare a sterile 50% glycerol/50% DEPC water solution.
?. Add 1 ml 50/50 solution to as many cryovials as you want, e.g. 6
?. Add 1 ml of bacterial culture to the cryovials, close them with the cap and shake them.
?. Store the cryovials in the -80 ° freezer.
TITLE: Protocol for derivatization and determination of structural monosaccharides in crude fungal exopolysaccharide
AUTHORS: tayebeh fooladi, Mohammad Reza Soudi, Majid M. Heravi, Sanaz Karimian

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify"></div><style>
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							</style></div><div class = "text-block"><span>Polysaccharides are, composed of monomeric simple sugars exhibiting substantial diversity for functional groups, found being suitable for vast biological activities in different living organisms. Difficulties in isolation, purification by professional methods and using costly equipment are the multiple challenges of characterizing the nature and properties of these important components of living cells. Several methods have been applied for the qualitative analysis of the carbohydrates. However, GC and GC-MS are the most appropriate techniques ever used to analyze complex structures of polysaccharide. Notably, so far, no commercial kit has been developed and offered, particularly for derivatization of polysaccharides. This study aims to confer a modified protocol for suitable derivatization of structurally unknown polysaccharides. The fungal exopolysaccharide from </span><span style = "font-style:italic;">Neopestalotiopsis</span><span> sp. has been used as the sample in this study. The study has established the multiple manual steps of the alditol acetate derivatization. The modifications have been highlighted as follows:</span></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;"> Trifluoroacetic acid (TFA) has been used instead of conventional trichloroacetic acid for hydrolyzing the crude exopolysaccharide. </li><li style = "counter-reset:ol0;list-style-type:disc;"> The standard sugars were treated separately, but not in the complex mixture.</li><li style = "counter-reset:ol0;list-style-type:disc;"> The simplicity of the method, using ordinary instruments and short time accomplishment of procedures, collectively makes this technique as the most applicable in a common research laboratory where no more sophisticated equipment is available.</li></ul></div></div>

[STEPS]
TITLE: Schedule of Events (Appendix 1 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 Appendix 1 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 participants with new-onset type 1 diabetes mellitus".</div></div>

[STEPS]
TITLE: Agarose gel pads for live Ashbya imaging
AUTHORS: ameya.jalihal 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A protocol to prepare agarose gel pads for live Ashbya imaging.</div></div>

[STEPS]
?. [Resuspension of Ashbya]
Make 20 mL 1X LFM stock before you startAlways check LFM tube for contaminationReconstitute with ddH2O
?. [Making agarose pads]
Depression slides live on Grace's desk. These are expensive, and we reuse them. Don't throw these out! Wash thoroughly with water and ethanol, dry them, and replace them in the box.
?. [Imaging]
?. [Making agarose pads]
Aliquot    of 1X LFM from step 1 into a 50 mL conical.
10 mL
?. [Making agarose pads]
Measure    agarose.
200 mg
?. [Making agarose pads]
Fill a 500 mL beaker with water to make a water bath and microwave the tube in  increments until the agarose is dissolved. Shake the tube to dissolve agarose.
?. [Making agarose pads]
Pipet  of the agarose solution into the depression on a depression slide.
200 µl
?. [Making agarose pads]
Drag a coverslip across the depression to create the pad.  After about 2-5 min, gently lift the coverslip off. The gel pad is now ready to use.
?. [Making agarose pads]
When ready, add  of the cells to the center of the pad. Gently cover it with a coverslip, and press with a kimwipe to squish the cells. Cells are now ready to image.
50 µl
?. [Resuspension of Ashbya]
Spin down overnight Ashbya culture to pellet cells in 15 mL conical tubes  300 RPM
?. [Resuspension of Ashbya]
Resuspend in 4 mL 1X LFM. Spin down again.   300 RPM
?. [Resuspension of Ashbya]
Resuspend in 1 mL 1X LFM. Incubate on the rotator at  until ready to make gel pad, or for .
30 °C
TITLE: Paintbrush method for C. elegans handling for microinjections
AUTHORS: Theresa V Gibney, Ariel M Pani

[DESCRIPTION]
See PDF attachment for protocol and links to videos.

This protocol describes a method for using a paintbrush to move C. elegans throughout the microinjection process. This method accelerates the microinjection process compared to traditional methods and improves the likelihood of worm survival. 

[STEPS]
TITLE: Protocol for Freezing in OCT
AUTHORS: Shin Lin, Yiing Lin

[STEPS]
?. Place tissue sample in culture dish with ice cold PBS.
?. Cut tissue piece with disposable razor blade.
?. In mold (choose mold depending on size of tissue), place ice cold OCT to cover the bottom.
?. Place tissue in mold atop OCT.  Fill mold with OCT until tissue is covered.
?. Place mold atop metal slab bathed in liquid nitrogen until sample is frozen.
?. Store sample in mold in -80ºC freezer.
TITLE: X-tremeGENE™ HP DNA Transfection Reagent Protocol for transfection of SH-SY5Y cells
AUTHORS: Laura  Smith

[DESCRIPTION]
SH-SY5Y cells were transfected with a pcDNA3.1 vector plasmid. Stable transfection was performed using the XtremeGENE reagent for 72 hours, and 400 µg/ml G418 antibiotics used as the selection marker . Colonies were selected and expanded for routine culture in growth media supplemented with G418. 

[STEPS]
1. Protocol from: https://www.sigmaaldrich.com/GB/en/technical-documents/protocol/cell-culture-and-cell-culture-analysis/transfection-and-gene-editing/xtghp-general-protocol
SECTION: Cell Preparation for Transfection
2. Plate SH-SY5Y cells in 10mm2dish approximately 24 hours before transfection, making sure cells are at optimal concentration (70–90 % confluency).
SECTION: Transfection
3. Allow      X-tremeGENE™ HP DNA Transfection Reagent, DNA and diluent (Opti:MEM®I      Reduced Serum Medium or serum-free medium) to warm to +15 °C to +25 °C,      and vortex gently.
Place      diluent in a sterile tube.
Add      plasmid DNA (2 µg) . Gently pipette up and down to mix.
Add      2 µL X-tremeGENE™ HP DNA Transfection Reagent to the diluted DNA.
Vortex      the mixture.
Incubate      for 15 minutes at +15 °C to +25 °C.
Add      transfection complex to the cells in a dropwise manner.
Gently      shake or swirl the wells or flasks to ensure even distribution over the  entire plate.
Incubate      cells for 72 hours before replacing with complete SH-SY5Y culture media (dx.doi.org/10.17504/protocols.io.bp2l617jzvqe/v1) supplemented with 400 µg/ml      G418 antibiotics and incubated at 37°C and 5% CO2 for selection.
SECTION: Culturing transfected SH-SY5Y cells
4. Growth medium with selection reagent was replaced every 2-3 days. 
After ensuring all mock transfections had died, colonies from the transfected cells were selected with sterile cloning cylinders. 
Colonies were transferred to 6 well plates and maintain in growth medium containing G418. 
Once confluent, cells were trypsinised and expanded into a 10mm2dish in 10 mL normal growth media. 
Clones were frozen down or pelleted for characterisation.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>A protocol for lithium acetate transformation of yeast that can be used to generate highly complex plasmid libraries (O(1E8) if starting with ~1L of culture) and can get over 1 million transformants from a single transformation (as described here).  Optimized using yeast strain Y8203.  Based on a protocol originally by Supipi Kaluarachchi Duffy.</p>

[STEPS]
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TITLE: Visualization of DNA of low concentration and molecular weight (DNA gel stain)
AUTHORS: Nadine   Mowoh, Stephane  Fadanka

[DESCRIPTION]
After PCR amplification of DNA, an agarose gel electrophoresis is run to separate the DNA based on their size.
The agarose gel consists of microscopic pores that act as a molecular sieve which separates molecules based upon the charge, size and shape. Agarose gel electrophoresis can also be used to separate other charged biomolecules such as RNA and proteins. Agarose is isolated from the seaweed genera Gelidium and Gracilaria and consists of repeated agarobiose (L- and D-galactose) subunits. The concentration of agarose in a gel depends on the sizes of the DNA fragments to be separated, with most gels ranging between 0.5%-2%. 

This protocol describes the steps in using the agarose gel electrophoresis to show that the BenBio DNA gel stain can allow the visualization of DNA of low molecular weight and concentration, and therefore good alternative to EtBr based DNA gel stains.

[BEFORE_START]
Ensure all materials, equipment and chemicals to be used for this experiment are all in place before starting. 

[GUIDELINES]
This procedure can be performed by laboratory staff that have been trained and have theoretical and practical skills in good laboratory practices. It can also be performed by molecular biology students or students of related fields under the supervision of a laboratory staff.

[STEPS]
SECTION: Visualization of DNA of low concentration and molecular weight
1. 
 

SECTION: Visualization of DNA of low concentration and molecular weight
2. To confirm visualization of DNA of low molecular weight
DNA gel stain

Follow the steps in preparing a 2% agarose gel as described in this protocol, 
We use the TO-DMSO DNA gel stain as "test DNA gel stain" and an EtBr based DNA gel stain as "positive control or standard DNA gel stain" (meaning 2 gels will be prepared). 
Load the gels (in this case a loading dye would not be used since the amplicon is a 50 or 100bp DNA ladder) therefore it can be loaded without loading dye needing 3-5ul of the ladder. Load directly into the gel wells in replicates.
Run the 2 gels simultaneously under the same conditions to finish and visualize the resulting band separation to confirm separation of the ladder into visible distinct bands (from the lowest to highest base pair bands) using a UV transilluminator or blue gel system.

To confirm visualization of a low concentration DNA

Make 1:5 serial dilutions of the 100bp DNA ladder or any DNA amplicon and use the different dilutions as amplicons to load a gel well.
Allow the gel to run through and visualize to determine the lowest concentration detectable by the gel stain.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is designed for 'around-the-horn' or 'divergent' PCR, where primers go around most or all of a plasmid but are pointed away from each other so they generate a linear product. Note that this protocol is written for Q5 polymerase, but works fine with other polymerases. To switch polymerases, just change the PCR reaction setup.</p>

[BEFORE_START]
<p>Program the thermocycler with the PCR program in the main protocol</p>

[STEPS]
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TITLE: ATP synthase activity assay (radioactive)
AUTHORS: Anika Wiegard, Christin Köbler, Katsuaki Oyama, Anja K. Dörrich, Chihiro Azai, Kazuki Terauchi, Annegret Wilde, Ilka Maria Axmann

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes how to detect synthesis of [α</span><span style = "vertical-align:super;">32</span><span>P]ATP from [α</span><span style = "vertical-align:super;">32</span><span>P]ATP by recombinant KaiC proteins. Radioactive nucleotides are separated via thin layer chromatography using TLC PEI Cellulose F plates as stationary phase and LiCl as soluble phase. The principle of this method is based on Egli </span><span style = "font-style:italic;">et al.</span><span> (Egli M, Mori T, Pattanayek R, Xu Y, Qin X, Johnson CH.2012. Dephosphorylation of the core clock protein KaiC in the cyanobacterial KaiABC circadian oscillator proceeds via an ATP synthase mechanism. Biochemistry 51:1547-58.)</span></div></div>

[STEPS]
?. [preparation]
express and purify KaiCoptional: phosphorylate or dephosphorylate KaiC prior to analysis
?. [incubation]
prepare a 25 µl mastermix with 3 μM KaiC in ATP synthesis buffer (20 mM Tris/HCl (pH8), 150 mM NaCl, 0.5 mM EDTA, 5 mM MgCl2, 0.5 mM ATP)add 2 ul [α-32P]ADP (= 20 μCi) freeze one 10 µl aliquot at -20 °C (0h, -20 °C)incubate one 10 µl aliquot for 2 hours at 30 °Cas a control, perform the same reaction but in the presence of 0.5 mM (non-radioactive) ADPin total you will have 4 samples for every KaiC protein of interest:  ABC1sample0.5 mM (non-radioactive) ADPincubation for 2h at2control 0h+-20 °C3control 2h+30 °C4sample 0h--20 °C5sample 2h-30 °C
ABC1sample0.5 mM (non-radioactive) ADPincubation for 2h at2control 0h+-20 °C3control 2h+30 °C4sample 0h--20 °C5sample 2h-30 °C
?. [thin layer chromatography]
spin down briefly and dilute samples 1:20 in H2Ospot  0.5 μl of the diluted reaction mixtures and  [α-32P]ADP and [γ-32P]ATP (as size controls) onto a TLC PEI Cellulose F plates (Merck Millipore) - place the spots approx 1-2 cm from the bottom of the plate, mark the level with a pencilwait until spots are driedpour a small amount of 1 M LiCl as mobile phase in a thin layer chromatography chamber (the solvent level must be lower than the distance of your spots from the bottom of the TLC plate)close the lid and wait a few minutesplace the TLC plate in the chamber, close the lid and allow the mobile phase to be drawn up the plate (without reaching the end of the TLC plate)remove and dry the TLC plate
?. [detection and analysis]
expose dried plates to an autoradiography screen and detect signals using for example a Personal Molecular Imager FX system (Bio-Rad)analyze the samples (for example with ImageLab software (Bio-Rad))for each lane determine the signal intensity of all detectable spotscalculate the relative intensity of [α-32P]ATP  as percentage of all signals in the corresponding lanefor normalization substract the relative [α-32P]ATP intensity of the -20 °C sample containing 0.5 mM ADP from the  relative [α-32P]ATP intensity in the other samples
TITLE: Nuclei Isolation from Human Meniscus for 10x Multiome
AUTHORS: Takuya Sakamoto, Erpei Wang, Merissa Olmer, Martin Lotz

[DESCRIPTION]
This protocol describes isolation of nuclei from fresh-frozen human knee meniscus for use in Omics analyses, including RNA-sequencing of ATAC-sequencing. Tissue dissociation was the most critical step in optimization which was monitored by nuclei yield, integrity and purity. Methods for quality control are also described.

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><span style="color: #000339; font-family: Raleway, sans-serif; font-size: 14px; font-style: normal; font-variant-ligatures: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: 2; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-stroke-width: 0px; background-color: #ffffff; display: inline !important; float: none;">This is a collection of CasPASE™ Apoptosis Fluorometic Assay protocols from G-Biosciences. Please follow the appropriate protocol, depending on your type of lysate.<br /></span></p>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Principle is that CTAB is charging the anionic nucleotides whereby neutral polysaccharides/ melanins are remaining in supernatant. This method also uses urea with the idea that the presence of urea helps to solubilize hydrophobic compounds that would otherwise potentially interact with the hydrophobic core of the CTAB micelles.

[STEPS]
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TITLE: Heart rate
AUTHORS: Núria Peñuelas

[DESCRIPTION]
Heart rate measurement in mice.

[STEPS]
1. Place the animal in mild anesthesia with isofluorane at 1.5% and 1l/min O2.
2. Prepare the PhysioSuite apparatus(Kent Scientific). Put the clip sensor in the right hind paw. Let it stabilize for 30 seconds before starting the recording.
3. When a stable heart rate is observed, perform 10 measurements.
4. Calculate the mean of the 10 measurements and represent the heart rate as average beats/minute.

represented as average beats/minute.
TITLE: Assessment of the in vitro trypanocidal activity
AUTHORS: Muhammad Muhsin Fathuddin

[DESCRIPTION]
The aim of this experiment is to assess the potential of a plant extract to fight off Trypanosoma species, including Trypanosoma brucei brucei, Trypanosoma evansi, etc., by testing it at concentrations ranging from 006.25 to 400.00 mg/mL. The results obtained indicate that the plant extract may be a useful tool in the management of trypanosomiasis, but further studies are required to confirm its effectiveness.

[BEFORE_START]
The experimental animals should be screened for any ailment at the commencement of the experiment using the laboratory’s recommended procedure and cleared of any ailment.

[GUIDELINES]
The experimental animals need to be handled in accordance with Good Laboratory Practices and Guidelines for Laboratory Animal Facilities and the recommendations of the Committee on Animal Use and Care

[STEPS]
SECTION: Antitrypansomal Activity
3. The infected rat to undergo euthanasia must have attained a blood parasitemia of log 8.4 or higher
 
.
SECTION: Antitrypansomal Activity
4. Euthanatized animal’s blood was dissolved in heparin (1 mL of heparin per 10 mL of blood) and mixed with glucose (0.1 g of glucose per 10 mL of blood).

SECTION: Antitrypansomal Activity
5. Then, aseptically, a clean micropipette transfers the blood (50 µL) to a clean, sterile microtiter plate into multiple wells.

SECTION: Antitrypansomal Activity
6. To the two wells containing blood, the same volume (i.e., 50 µL) of the highest drug concentration was added, respectively.

SECTION: Antitrypansomal Activity
8. The negative control blood was mixed with the dilutant used to prepare the various concentrations (i.e., dextrose-saline or water).
SECTION: Antitrypansomal Activity
9. The positive tests were also performed with the standard recommended concentrations
of a commercially available trypanocide drug.
SECTION: Antitrypansomal Activity
7. Step 4 was repeated for the other different concentrations available.
SECTION: Extract Preparation
1. To produce a stock   solution of100 mg/mL for the extract, weigh1 g (1000 mg) of the extract, which was solubilized in 1 mL of dimethylsulfoxide (DMSO) solution and made up to 9 mL in dextrose saline/normal saline.
SECTION: Extract Preparation
2. Serial dilutions were made for the remaining concentration of50 mg/mL, 25 mg/mL, 12.5 mg/mL  and 6.25 mg/mL of the extracts were made in Dextrose saline (freshly prepared).
SECTION: Antitrypansomal Activity
10. At 5 min  5-minute interval, a drop of blood is taken from each well and smeared on a clean glass slide. then observe under a microscope at 400x for signs of motility unique to Trypanosoma spp.
 

 


SECTION: Antitrypansomal Activity
11. The above step is repeated with each well. Till motility stopped in all wells
TITLE: T-RFLP Sephadex purification and sample preparation
AUTHORS: Eva Petrova, Roey Angel

[DESCRIPTION]
Purification of PCR-product after restriction digest. Samples proceeded on the capillary sequencer.

[BEFORE_START]
Materials:
 
digested PCR-product (100-200ng in 20ul)
96 well filter plates
96 well collection plates (IBL 96*0.2ml frosted Subkirted thin well)
Sephadex G50 (GE, 37.5g into 500ml autoclaved MilliQ H2O)
Multipette 10ml/1ml
Multichanel 200ul/10ul
HiDi Formamide (Prepare a master mix: 10ul HiDi Formamide + 0.3ul Size Standard, GS_1200 LIZ, AB per sample nebo 165ul HiDi Formamide + 5ul Size Standard for one run = 16 samples)

[GUIDELINES]
The Sephadex solution is stored in a glass bottle in the fridge. Before you start the purification let the Sephadex solution come to room temperature and shake well before each usage!

[STEPS]
SECTION: Sephadex purification
1. Load 200μl Sephadex solution onto the 96 well filter plates (shake the Sephadex solution well before each usage!) (use the EppendorfMultipette with a 10ml tip), the no. of wells you load depends on the no. of samples,
load filter plate on 96 well collection plate, 
place the lit on top of this 'plate sandwich' and
place in swing out rotor base.
200 1
SECTION: Sephadex purification
2. Spin at 230rcf(g) for 1 min 15sec (Eppendorf centrifuge 5810). Don't forget to balance the centrifuge by placing a dummy  'plate sandwich'  on the rotor.
 
1 min
SECTION: Sephadex purification
3. Discard flow through.
SECTION: Sephadex purification
4. Repeat step 2. (Shake Sephadex!)
   
SECTION: Sephadex purification
5. Place Sephadex G-50 containing 96 well filter plate on the collection plate.
SECTION: Sephadex purification
6. Load 20μl sample in the centre of the Sephadex column. Just drop the 20ul on tj top of the gel. Avoid to destroy the Sd. gel by penetrating it  with the pipette tip.
20 1
SECTION: Sephadex purification
10. Go immediately to sample preparation or store the samples at -20°C.
SECTION: Sephadex purification
7. Spin at 420 rcf (g) for 1 min (Eppendorf centrifuge 5810).  Note: There will be some extra buffer that comes through from the Sephadex resulting in a total sample volume of ∼ 35-40 μl.
1 min
 
SECTION: Sephadex purification
9. Remove the Sephadex from the filter plate and rinse the filter plate and collection plate as well (with MQ). Both plates could be reused several times.
SECTION: Sephadex purification
8. Transfer the purified samples back to the original caps.
SECTION: Sample preparation for capillary sequencing
11. Aliquot 10μl of HiDi Formamide master mix into a new 96 well plate.
SECTION: Sample preparation for capillary sequencing
12. Add the 4μl of your purified sample per well.
4 1
SECTION: Sample preparation for capillary sequencing
13. Denature samples for 3min at 95°C.
3 min
95 10
SECTION: Sample preparation for capillary sequencing
14. Transfer samples immediately on ice and let the chill for 5 min.
0 10
5 min
SECTION: Sample preparation for capillary sequencing
15. Store samples in the fridge, prepare the sample sheet and run the samples at ABI 3130XL Capillary Sequencer.