eamag/protocols-with-steps huggingface dataset:
TITLE: PROTAC Design based on Bioinformatics
AUTHORS: boc.protac  

[DESCRIPTION]
In order to simplify and accelerate the discovery of PROTAC, it is necessary to grasp the design rules of PROTAC, which is helpful to establish a reliable PROTAC evaluation platform. To date, most reported PROTAC designs are based on target protein-binding ligands (usually inhibitors). Because the crystal complex structure of the target protein and its binding ligand and the structure-activity relationship information of the ligand have been used to guide PROTAC design, such as determining the Linker binding site of the target protein binding ligand. Recent research uses bioinformatics to guide PROTAC optimization. Bioinformatics methods play an irreplaceable role in the process of drug discovery, especially for the analysis of large-scale multi-group data. at present, many disease-related database resources have emerged, and a variety of bioinformatics methods have been established to discover potential drug targets based on biological network characteristics, multi-gene chip, proteome, metabolome data and so on. The target drug availability and drug side effects were predicted.

As a leading service provider in drug discovery and research, BOC Sciences is fully capable and committed to providing one-stop proteolysis targeting molecular drug discovery based on chimeric (PROTAC®). With a comp rehensive and advanced platform, we provides PROTAC design based on bioinformaticsservices to customers around the world to meet new drug discovery goals.

[STEPS]
TITLE: Encasing LAMP mixture in paraffin
AUTHORS: Niels Appelman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol can be used to encase LAMP mastermix and primers in paraffin.</div></div>

[STEPS]
?. Prepare a 10X primermix according to the following scheme  AB1Primer  Concentration2 FIP 16 μM3 BIP 16 μM4 F3 2 μM5 B3 2 μM 6 LOOP F 8 μM 7 LOOP B8 μM
AB1Primer  Concentration2 FIP 16 μM3 BIP 16 μM4 F3 2 μM5 B3 2 μM 6 LOOP F 8 μM 7 LOOP B8 μM
?. Add the following components to a micro centrifuge tube  AB1  Component
    Volume added
  2  Mastermix
  5 ul3  Lamp
  primers (10X)
  1 ul
AB1  Component
    Volume added
  2  Mastermix
  5 ul3  Lamp
  primers (10X)
  1 ul
?. Open the centrifuge tube and let dry at 37 C until only solid is left.

Note: The longer this step takes, the more activity the enzyme loses. It is recommended to dry in a storage area with a low humidity, as to speed up this step. To minimize the loss of activity, lyophilisation is recommended.
?. Add 4 mg of paraffin wax
?. Heat at 60 C for 5 minutes to let the wax melt and encapsulate the dried LAMP mixture
?. After the wax dries, the continued activity of the enzyme can be tested. To do this, add the following components  AB1  Component
    Volume added
  2  Template
    x
  uL
  3  MilliQ
    10
  - x uL
AB1  Component
    Volume added
  2  Template
    x
  uL
  3  MilliQ
    10
  - x uL
?. Incubate at 65 C for 30 minutes
?. Record the color of all samples. Yellowing of a sample indicates a positive result.
?. In case of doubt regarding the result of a sample, run it on an agarose gel. The presence of many bands, often not individually recognizable, indicates a positive result.
TITLE: S-Basal Medium
AUTHORS: Adrien Assie, Buck Samuel

[DESCRIPTION]
Large quantities of C. elegans can be grown in a liquid medium. Liquid cultures of C. elegans are usually grown on S Medium using concentrated E. coliOP50 as a food source. This is the S-Basal Medium recipe

[STEPS]
1. Start with 700 mL

2. 5.9 g NaCl (100 millimolar (mM))


3. 50 mL of 1 Molarity (M) Potassium Phosphate Buffer, pH 6.0

OR

1 g and 6 g 

4. Adjust water to 1000 mL

5. Autoclave
TITLE: Lakes ABPS Protocol - Optimized protocol for the extraction of fish DNA from freshwater sediments (Thomson-Laing et al., 2022)
AUTHORS: Georgia Thomson-Laing

[DESCRIPTION]
DNA was extracted from lake sediment samples by an alkaline lysis method with ethanol precipitation adapted from method described by Kuwae et al. (2020); Sakata et al. (2020b); Sakata et al. (2020a). 

In a comparison of multiple sedDNA extraction methods, the ABPS (Alkaline buffer - power soil) protocol yielded the highest concentrations of target genets across a range of lake sediments. This protocol was further optimized (65C incubation temperature, pooling of multiple PowerSoil extractions) to overcome technical challenges related to co-precipitation of organic content in lake-surface sediments.

The optimized ABPS protocol is called the "Lakes ABPS protocol"

This protocol has proven to be successful at detecting fish sedDNA from surface sediments in multiple systems for multiple species.

[STEPS]
SECTION: Alkaline extraction
1. INTO a 50 mL tube add:
10 g of sediment sample
6 mL sodium hydroxide (0.33M)
3 mL Tris-EDTA (pH 8)
SECTION: Alkaline extraction
2. VORTEX for 1 min 

INCUBATE at 65 °C for 50 min 
SECTION: Alkaline extraction
3. ALLOW samples to cool to Room temperature 

CENTRIFUGE at 15000 x g for 60 min 
SECTION: Ethanol precipitation
4. TRANSFER 7.5 mL of supernatant to a new 50 mL tube

ADD 7.5 mL of Tris HCl (1M, pH 6.7) to neutralize
SECTION: Ethanol precipitation
5. ADD 1.5 mL sodium acetate (3M, pH 5.2)

ADD 30 mL of molecular grade 100% ethanol

INCUBATE samples at -20 °C for <60 min 
SECTION: Ethanol precipitation
6. CENTRIFUGE samples at 10000 x g for 60 min 

DISCARD supernatant

RETAIN precipitated pellet
SECTION: DNeasy PowerSoil extraction
7. EXTRACT the total pellet using multiple DNeasy PowerSoil DNA Isolation Kit extractions following the manufacturer's instructions
 

POOL resultant DNA elutes

DNA is now ready for downstream applications
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This procedure is suitable for very low to high concentration of phosphorus (0.00001- 6 mg/l).</p>
<p style="text-align: justify;"><br /><strong>What happens to this procedure</strong>: Ammonium molybdate and potassium antimonyl tartrate react in acidic solution with orthophosphate to form a heteropolyic acid (phosphomopydbic acid) which can be reduced by ascorbic acid to form an intense blue color, the absorbance of which is then measured at 885 nm spectrums.</p>
<p style="text-align: justify;"><br /><strong>Forms of Phosphorus:</strong> In the natural water phosphorus exists solely in three basic forms: orthophosphates, condensed phosphates (pyro-, meta- and polyphosphates) and organically bound phosphate. All of which combined together to form total phosphorus. From the sample water, orthophosphate can be analyzed directly by spectrophotometry. But for measuring total phosphorus-phosphate, condensed phosphate and, organically bound phosphate need to be digested to orthophosphate prior to the final procedure.</p>

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

[DESCRIPTION]
<p>Adapted from: Van Etten, J. (n.d.). Titering of <em>Chlorella </em>Viruses. Retrieved from http://ncv.unl.edu/vanettenlab/</p>

[GUIDELINES]
<p>For critical work, a second purification may be necessary.</p>
<p> </p>
<div>
<div>
<p>This protocol is used for Chloroviruses and may need to be optimized according to the system that you are working with. Make sure that the virus sample that you are using will pellet sufficiently at this RPM in the ultracentrifuge before using this protocol.</p>
</div>
</div>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Streptavidin yielding with Ponceau
AUTHORS: Manuela de las Casas

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The aim is to see if the streptavidin binds correctly to the nitrocelullose membrane. Only after achieving a positive result is recommended to proceed to the next step (the dot blot).</div></div>

[STEPS]
?. [Preparing the nitrocellulose strip]
Set the hot plate to 100ºC or at least warm enough to melt the wax. Cut a small amount from one of the wax pencils and place it  on a Petri dish. Set the Petri dish on the hot plate and wait for the wax to melt. Cut a strip from the nitrocellulose sheet with the desired size.Once the wax is melt, grab a 200μL pipette tip from the pointy side and place the broad end in the wax. Check the whole circle contains enough wax and carefully place it on the nitrocellulose strip. Apply a little pressure and remove the pipette tip. You should be able to see the wax circle on the strip.Cover the nitrocellulose strip with paper (one sheet on top, another underneath). Place the sandwich on the hot place with a Petri dish on top (this is just to apply some pressure, any temperature-resistant flat object is valid here). Allow the strip to warm for 5 minutes. This will allow the wax to penetrate all through the strip.Remove the strip from the hot plat and let it cool down again. You should see a circle of wax but the center must be clear, otherwise the protein will not have anywhere to bind. If the circle is too thick, consider repeating these steps.
?. [Adding the streptavidin]
Pipette 5 μL of Streptavidin. If the protein is too diluted, you might not see a staining with the Ponceau. We ended using the protein suspended in PBS but without any dilutions. Allow it to dry completely. Room temperature is adequate.
?. [Ponceau yielding]
Set the strip on a Petri dish and let it soak completely in Ponceau stain. Wash it repeatedly with water until the edges of the strip (where is no protein) are clear. Let it dry. A positive result should show a difference in color between inside and outside the wax circle. The more concentrated the protein, the more intense should be the staining.
TITLE: Restriction Enzyme Digestion (Protocol for NEB CutSmart® Buffer)
AUTHORS: Alba Balletbó

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used to check if the two selected restriction enzymes can perform effective catalysis in the same solution.</div></div>

[STEPS]
?. Mix DNA solution with the suitable amount of the master mix. Reaction system for a total volume of 25μL:  AB1DNA solution20 μL210x NEB CutSmart® Buffer2.75 μL3Restriction enzyme1.0  μL4ddH2O1.25 μL5Total Volume25 μL
AB1DNA solution20 μL210x NEB CutSmart® Buffer2.75 μL3Restriction enzyme1.0  μL4ddH2O1.25 μL5Total Volume25 μL
?. Mix gently in the eppendorf tube.
?. Incubate at 37ºC for 20 minutes.
TITLE: Optogenetic modulation of dopaminergic neurons
AUTHORS: Cristian González-Cabrera, Matthias Prigge

[DESCRIPTION]
This protocol describes an optogenetic activation of neuromelanine-laden dopaminergic neurons in the SNc-VTA. We induce neuromelanin (NM) in the SNc-VTA through viral expression of a humane tyrosinase virus. Furhtermore we express a red-absorbing opsin in one hemispherend and blue absorbing in the other hemisphere. We detail how we optognetic stimulateanimals in over four weeks.

THis optogenetic deep brain stimulation induces a reduction in neuromelanine levels in dopaminergic neurons, and rescues behavioral phenotypes as evaluate in the grip strength test.

[STEPS]
SECTION: Viral Injection
1. Stereotactic injection of viruses into DAT-Cre (JAX# 006660) animals

Viral Constructs:
1.- pAAV-hSyn1-dlox-ChrimsonR_tdTomato(rev)-dlox-WPRE 

2.- pAAV-hSyn1-dlox-hCHR2(H134R)_mcherry(rev)-dlox-WPRE

3.- pAAV_Ef1a_DIO_hTyrHA_minBack

Injections: 
- Bilateral injections were performed. 
- Right hemisphere: viruses 1 and 3. 
- Left hemisphere: viruses 2 and 3.
- Viruses were combined 50:50 and injected a total Volume of 600ul per hemisphere

Steroetactic coordinates:
Subtantia nigra

ML: 1.4mm AP: 3.25mm DV: 4.0 (blunt needle / NF34BL-2)

Injection set-up:
Hamiliton syringe couple t oa WPI injector (UMP3T-1) (link) 
Injection speed 100nl/min
SECTION: Experimental Timeline
2. - Animals were left to accumulate neuromelanin for 6 or 10 weeks.
- Animals were stimulated for 30 minutes for 4 weeks. Stimulation during 5 days and two days rest.
- The stimulation was 635nm, 5ms pulse, 10 Hz at at 10mW. 3 seconds ON 10 seconds OFF.
SECTION: Optical stimulation
3. fibers are attached bilaterally to the animals
the animal is placed in open field arena
stimulation is controlled via PulsePal (OpenEphys link) on a MRL_III-635L laser
TITLE: 10x Multiome Sample Processing
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
Protocol is used to profile all the open chromatin regions at a single nuclei level through the rapid generation of NGS-ready libraries, and for rapid generation of 3' transcriptomic-NGS-ready-single-cell-libraries from the same pool of transposed nuclei.

"Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Numbers 1U01MH114812-01 and 1U19MH114830-01. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health".

[STEPS]
TITLE: Beneficial Bio Products: Packaging protocol
AUTHORS: Nadine    Mowoh, Stephane  Fadanka

[DESCRIPTION]
After production and QC (internal and External) of our products, some documents are prepared which will accompany the reagent or products whenever they are sent to a user to ensure they are used correctly to avoid altered results. 
There include the Product Specification sheet which carries information on the reagent description, composition, use and storage. 
Another important document is the Certificate of Analysis with a list of all QC tests that have been performed on the reagent and the Datasheet which contains reagent features, application, protocol for use

[BEFORE_START]
Make sure all materials and equipment needed for packaging are available and functional.
Ensure that any documents to accompany the product are also printed and included in the packaging.

[GUIDELINES]
Wear protective clothing and practice aseptic techniques during packaging to reduce or control contamination that may affect the efficacy of the products.

[STEPS]
SECTION: Gather all the information about the order at hand or product to be packaged
1. The information include:
Nature and quantity of products; Destination; Customers detail; Date and expected delivery date…). This information will guide on the type of packaging that will be required.
 

SECTION: Print specific amounts and type of labels and stickers
2. Current equipment used to print sticker and label: 
Labelwriter 450 Turbo from Dymo
Dymo 54 mm*101mm 220 Label
SKU: ED1F-073WH Cryogenic Labels (0.7’’x1.49’’) 
SKU: 30252 LW Address Labels (1, 1/8" x 3, 1/2")
Once set Up and installed, use the Dymo label printer to print suitable products and customer information stickers( print the corresponding sticker with product name, date of manufacture, expiration date, storage condition etc.)
Pre-designed (Ready-made) versions of product labels and customer information labels can be made and Information on the design can be modified to fit new products. 
Choose the appropriate packaging (Size of the envelope, size of the plastic bag…)depending on the product to be delivered
SECTION: Packaging
3. Follow the packaging chain; 

 

Unit 1: Inner package completion

Place the appropriate product sticker on the corresponding product
 Place the product inside the appropriate plastic sachet;
Seal the sachet;

Unit 2: Outer package completion

Fold the Datasheet and the Welcome message appropriately;
Place the plastic sachet containing the product inside the padded envelope;
Place the folded datasheet and welcome message in the envelope; 
Seal the envelope;
Stick a BenBio Logo on the external side of the envelope;
Stick the customer information sticker on the opening side of the envelope;

Unit 3: Shipping package

Place the padded envelope inside an A4 envelope;
Seal the envelope and add customers' contact and address information.
TITLE: Diaminobenzidine (DAB) Staining
AUTHORS: Haley   Geertsma

[DESCRIPTION]
This protocol is used to stain paraffin-embedded mouse brain tissue.

[STEPS]
1. To paraffin-embedded tissue, deparaffinize in 100% xylenes for 3x 5 minutes.
2. Incubate in 100% ethanol for 2x 5 minutes.
3. Incubate in 70% ethanol for 5 minutes.
4. Incubate in 50% ethanol for 5 minutes.
5. Incubate in 1X PBS for 5 minutes.
6. Perform antigen retrieval by incubating tissue in 1X sodium citrate for 20 minutes at 95oC.
Sodium citrate: 10mM sodium citrate + 0.05% Tween-20, pH6
7. Wash with 1X PBS for 5 minutes.
8. Incubate in 0.9% H2O2 for 10 minutes at room temperature.
9. Wash with 1X PBS for 5 minutes.
10. Incubate in blocking buffer for 1 hour at room temperature.
Blocking buffer: 10% serum + 0.05% Triton X-100 in 1X PBS
11. Wash with 1X PBS for 5 minutes.
12. Incubate in primary antibody diluted in blocking buffer overnight at 4oC.
13. Wash with 1X PBS for 5x 5 minutes.
14. Incubate in secondary antibody diluted in blocking buffer for 1 hour at room temperature.
15. Wash with 1X PBS for 5x 5 minutes.
16. Incubate in streptavidin horseradish peroxidase diluted in 1X PBS for 1 hour at room temperature.
17. Wash with 1X PBS for 5x 5 minutes.
18. Expose to DAB for 3-10 minutes then rinse in 1X PBS for 2x 2 minutes.
19. Incubate in 50% ethanol for 2 minutes.
20. Incubate in 70% ethanol for 2 minutes.
21. Incubate in 100% ethanol for 2 minutes.
22. Incubate in 100% xylenes for 5 minutes.
23. Coverslip with Permount and a #1.5 coverslip.
TITLE: Proton Density Fat Fraction (PDFF) Measurement of Myelofibrosis in Mouse Tibia
AUTHORS: Thomas L Chenevert, Thomas Chenevert

[DESCRIPTION]
This document details procedures for PDFF measurement in MF mouse tibia to achieve stated performance claims.In this profile PDFF values are expressed in “% units” on a 0 to 100% scale, such that bone marrow containing dominant red marrow and dominant yellow marrow have PDFF values of 0-few% and 80-100%, respectively. Tibia bone marrow composition in MF mouse models has gradation going from proximal to distil ends of the tibia, therefore separate claims are made for volume of interest (VOI) analysis of PDFF maps for each of three distinct sections along the length of the tibia (see Figure 1):
Section 1 (proximal)º VOI (~4-5mm3) within 9mm of proximal end of tibia
Section 2 (transition)º VOI (~0.4-0.5mm3) from 10 to 12mm of proximal end of tibia
Section 3 (distil)º VOI (~0.1-0.2mm3) from 13 to 14mm of proximal end of tibia

Claim 1:A measured change in the mean PDFF in Section 1 VOI of MF mouse model tibia that exceeds ±1.6% indicates a true biological change has occurred in the tibia bone marrow with 95% confidence.
Claim 2:A measured change in the mean PDFF in Section 2 VOI of MF mouse model tibia that exceeds ±15.5% indicates a true biological change has occurred in the tibia bone marrow with 95% confidence
Claim 3:A measured change in the mean PDFF in Section 3 VOI of MF mouse model tibia that exceeds ±25.5% indicates a true biological change has occurred in the tibia bone marrow with 95% confidence

[STEPS]
TITLE: 10x Protocols: Chromium Single Cell/Nuclei Gene Expression Flex Multiplex-- University of Minnesota TMCs (CG000527 Rev C)
AUTHORS: 10x Genomics

[DESCRIPTION]
10x Genomics Chromium Single Cell Expression flex protocol for library construction. 

Protocol ID# (CG) and Revision letter provided here: 
    10x protocol CG000527, Revision C -- Library construction of 10x fixed single cell or nuclei

Note: These protocols may not be the current version offered by the company but were used to produce the specific datasets connected to them. Please review the company support websites for the most recent versions of the protocols prior to starting your experiment. 

[STEPS]
1. https://www.10xgenomics.com/products/single-cell-gene-expression-flex
https://www.10xgenomics.com/support/single-cell-gene-expression-flex
2. Complete single cell or nuclei isolation and 10x fixation prior to starting this protocol
3. 10x Protocol CG000527, Revision C (Library Construction):
TITLE: DNA extraction - Zooplankton - 50 tubes
AUTHORS: coline.royaux, Nicolas Rabet, Céline Bonillo, Myriam Georges

[DESCRIPTION]
This protocol is derived from the QIAamp DNA micro kit (QIAGEN) protocol and was used to extract DNA from whole or parts of zooplanktonic freshwater crustaceans (Copepoda, Branchiopoda, ...) from New Caledonia.

[BEFORE_START]
Make sure to properly prepare your buffers (e.g. heating, adding ethanol)

[STEPS]
1. Prepare your 50 tubes with one individual per well. Alternate genus in the wells to detect eventual contamination between wells.
1.1. Collect one individual from a sample
1.2. Note its genus and determine its sex with a binocular microscope
1.3. For big individuals (more than 5 mm), dissect a few legs and put it in the well. Be careful not to damage the rest of the body and put it in a tagged Eppendorf tube.
For little individuals (less than 5 mm), put the whole body.
 

1.4. When all tubes are filled, the biological material has to dry before going to lysis

 

 



2. Prepare the lysis
2.1. Add 180 µL  in each well and equilibrate to room temperature

3. Put your tubes in a proofer at 56 °C    to lyse the tissues
4. Perform the DNA extraction manually

 

4.2. Add 200 µL in each tube and mix by pulse-vortexing for 15 s 

4.7. Add 500 µL in the column, centrifuge 8000 rpm, 1 min , discard the collection tube containing the flow through and place the column in a clean 2mL collection tube.

4.8. Add 500 µL in the column, centrifuge 8000 rpm, 1 min , discard the collection tube containing the flow through and place the column in a clean 2mL collection tube.
4.9. Centrifuge 3 min to dry the membrane completely.

4.10. Place the column in a clean, closable, eppendorf tube.

2.2. Add 20 µLin each well, mix by pulse-vortexing for 15 s and briefly centrifuge

4.3. Add 200 µL in each tube, mix by pulse-vortexing for 15 s , incubate at room temperature for 5 min.

4.1. Optional steps, if RNA carrier is needed : Quantity for twenty tubes (using 20 volumes + 2 margin to avoid errors = 22 volumes)
Mix 22 µg  with 22 µL and mix until dissolved.
Mix 22 µL with 4400 µL (200 µL and mix.

4.4. Briefly centrifuge to remove drops from the lid.
4.5. Prepare all the eluting columns by puting each in a 2mL collection tube.
4.6. Transfer the prepared DNA lysate into the eluting column with a pipette, centrifuge the column 8000 rpm, 1 min , discard the collection tube containing the flow through and place the column in a clean 2mL collection tube.

 


4.11. Add 50 µL in the column, incubate at room temperature for 1 min and centrifuge 1 min. The DNA extract in the clean eppendorf tube, discard the column.
TITLE: Digestion with NEBNext dsDNA Fragmentase (M0348)
AUTHORS: New England   Biolabs

[DESCRIPTION]
NEBNext dsDNA Fragmentase is an enzyme-based reagent that shears DNA to produce fragments of the desired sizes in a time-dependent manner, for next generation sequencing library preparation protocols
dsDNA Fragmentase provides random fragmentation, similar to mechanical methods (1,2).

[BEFORE_START]
Adequate mixing of NEBNext dsDNA Fragmentase is important for the success of this reaction. NEBNext dsDNA Fragmentase should be vortexed for 3 s prior to use.

For tough digestions, add 1 µL of 200 millimolar (mM) to the reaction. Additional MgCl2 can be added if necessary.

The protocol listed below is for fragmentation of 5 ng–3 μg of DNA.

[STEPS]
2. Combine the following components in a sterile PCR tube and vortex: 
   component amount   DNA (5 ng–3 μg) 1–16 μl   10X Fragmentase Reaction Buffer v2 2 μl   Sterile Water  variable

3. Add 2 µL and vortex mixture for 3 s.
 

4. Incubate at 37 °C for the recommended times below to generate the desired fragment size:
 

   Desired Fragment Size (bp) Incubation Time (min)   50–200 25–35   200–1,000 15–25   1,000–2,000 10–15   

 

5. Add 5 µL  to stop the reaction.
6. Clean up the fragmented DNA with column purification or using SPRI beads.
 

For further analysis:

Bioanalyzer: Clean up the fragmented DNA prior to loading on a Bioanalyzer chip. 
End Repair: Clean up the fragmented DNA then proceed with desired DNA end repair protocol.
Polyacrylamide Gel Analysis: Clean up the fragmented DNA prior to loading the samples on a PAGE gel.
Long Term Storage: Clean up the fragmented DNA prior to long term storage.
Agarose Gel Size Selection/Analysis: Samples can be loaded directly on to an agarose gel. It is not necessary to clean up the reactions prior to loading.
1. Vortex NEBNext dsDNA Fragmentase for 3 s, quick spin and place on ice.