eamag/protocols-with-steps huggingface dataset:
TITLE: Cytochrome C Assay_small_volume
AUTHORS: Elizabeth Fozo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Cytochrome C Assay --- Small volume</div></div>

[STEPS]
?. [Cytochrome C Assay -- Small volume]
Start ON cultures of the strain of interest
?. [Cytochrome C Assay -- Small volume]
The next day, measure OD600 and calculate how much of your overnight culture you need for OD600 ~ 0.01
?. [Cytochrome C Assay -- Small volume]
Add supplement if doing Long Term exposure or place BHI + 0.01 cells directly into 37oC for a later spike-in.
?. [Cytochrome C Assay -- Small volume]
Harvest cells at 0.3 if doing Long Term or 0.225 - 0.25 if doing Spike
?. [Cytochrome C Assay -- Small volume]
Obtain OD600 values fro each of your cultures and then calculate how much of your culture is needed to reach OD600 of 7You need 1mL cells at OD600 of 7Formula: (7 - OD600)(1mL) = (measured log phase- OD)(X mL)You will need around 25mL to achieve this concentration
?. [Cytochrome C Assay -- Small volume]
Use a 50 mL conical and centrifuge your cells for ~ 10 minutes at 3500 RPM. Pour off supernatant
?. [Cytochrome C Assay -- Small volume]
Wash the cells with 10 mL of 20mM MOPs buffer*(stock is 1M) by centrifugation for 5 minutes at 3500 RPM. 20 mM MOPS buffer is made in H2O.
?. [Cytochrome C Assay -- Small volume]
Repeat the wash
?. [Cytochrome C Assay -- Small volume]
Resuspend the pellet in 1 mL of 20 mM MOPS buffer
?. [Cytochrome C Assay -- Small volume]
Add 1mg/mL of Cytochrome C (stock is 10mg/mL in HO) - vortex
?. [Cytochrome C Assay -- Small volume]
Incubate for 10 minutes at RT
?. [Cytochrome C Assay -- Small volume]
Centrifuge for 10 minutes at 3500 RPM
?. [Cytochrome C Assay -- Small volume]
Add 800uL of the supernatant to plastic cuvette
?. [Cytochrome C Assay -- Small volume]
While cytochrome exposed cells are spinning, make standards. To 1 mL of 20mM MOPS buffer add(remove the appropriate volume of liquid from each 1 mL):0.1 = 10uL of cytochrome0.2 = 20uL of cytochrome0.3 = 30uL of cytochrome0.4 = 40uL of cytochrome
?. [Cytochrome C Assay -- Small volume]
Add 800uL of each standard
?. [Cytochrome C Assay -- Small volume]
Use the Cytochrome program ("CYTOCHROME") on the Fozo Lab Spec = OD530
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Preparing data for use in vContact by using VirSorted <a href="https://github.com/MicroB3-IS/osd-analysis" target="_blank">Ocean Sampling Day (2014)</a> contigs, using tools available in <a href="https://www.protocols.io/view/cyverse.org" target="_blank">Cyverse</a>. This protocol creates a BLAST DB, BLASTs sequences, and creates a gene-to-contig mapping file. Results from this protocol are suitable for vContact-PCs.

[BEFORE_START]
To run this protocol, users must first <a href="https://user.iplantcollaborative.org/register/" target="_blank">register</a> for Cyverse account. All data (both inputs and outputs) are available within Cyverse's data store at /iplant/home/shared/iVirus/ExampleData/

[GUIDELINES]
This is part of a larger protocol <em>Collection</em> that involves the end-to-end processing of raw viral metagenomic reads obtained from a sequencing facility to assembly and analysis using Apps (i.e. tools) developed by iVirus and implemented within the Cyverse cyberinfrastructure.

[STEPS]
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TITLE: Cell Penetrating Peptide Design and Synthesis
AUTHORS: Cathy  Miller

[DESCRIPTION]
Cell penetrating peptides (CPPs), also known as protein transduction domains (PTDs) or membrane transduction peptides (MTPs), are small molecular peptides that consist of 5-30 amino acid residues, which not only penetrate the cell membrane by themselves, but also carry a variety of exogenous substances such as peptides, proteins, nucleic acids and nanoparticles in a covalent or non-covalent binding manner into cells. CPPs have a wide range of transmembrane targets and can enter a variety of cell types, even through the blood-brain barrier when appliedin vivo.


As an effective bioactive molecular intracellular transport tool, CPPs have broad application prospects in cell biology and cellular immunology, especially in the fields of drug development, gene biotherapy and tumor targeted therapy.

Creative Peptides has focused on the design and synthesis of peptides for many years. With state-of-the-art equipment and experienced chemists, we can provide customers with GMP-grade cell-penetrating peptide synthesis services such as HIV-TAT and Oligo-Arg, etc. At the same time, we can also design new cell penetrating peptides with unsuitable or non-natural amino acids, peptide branches and other modifications according to customer needs to design suitable cell penetrating peptides to meet the research needs of customers. Creative Peptides offers a comprehensive series of cell-penetrating peptides complying with specific purities, scales and modifications for pharmaceutical and biochemical applications.

[STEPS]
TITLE: ADBS Whole Genome Sequencing (WGS) analysis pipeline for Genomic-QC Report
AUTHORS: Ravi More

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Whole Genome Sequencing (WGS) analysis pipeline devloped for generating Genomic-QC Report in Accelerator   Program  for  Discovery  in  Brain  Disorders   Using  Stem   Cells   (ADBS) program.</div></div>

[STEPS]
?. [Define paths and directories]
?. [Unzip the raw reads files from .gz to fastq format]
?. [QC check of R1 and R2 paired-end raw reads using FASTQC, Trimming poor quality reads using Prinseq-lite, and Adapter contimination removal using AfterQC]
Software versions used: FASTQC version 0.10.1 Prinseq-lite version 0.20.4AfterQC version 0.9.6
?. [Alignment of clened raw reads against Human Reference Genome hg19 GRCh37.p13 build using BWA and SAMTOOLS.]
BWA version 0.5.9Samtools version 1.3.1
?. [Mark PCR duplicates and sorting BAM using PICARD Tools]
Picard version 2.0.1Samtools version 1.3.1
?. [INDEL re-alignment using GATK tools]
GATK version 3.6
?. [SNP and INDEL variant calling using Isaac Variant Caller tool and filter  SNP and INDEL using rtg-tools]
Isaac Variant Caller -- 1.0.7rtg-tools version  3.7.1
?. [Check the alignment QC of the bam file using Qualimap]
Qualimap version 2.2.1
?. [VCF QC of SNP and INDEL files using rtg-tools]
rtg-tools version 3.7.1
?. [SNP AND INDEL variant annotation using ANNOVAR]
ANNOVAR reference assembly 65 with reference hg19
?. [Mitochondria analysis]
Extracting mitochondrial reads from BAM file and creatiing anather BAM file to input mtDNA-Server tool for Mitochondria analysisSamtools version 1.3
?. [Blood Group Prediction]
BOOGIE - Phenotype prediction from NGS data Version: 1.0
?. [SNP-Chip rsID comparison with WGS rsID]
?. [Extract Damaging Varaints (SIFT, PolyPhen) from SNP file]
?. [HLA Analysis using HLAVBSeq]
Read data aligned to GRCh37/hg19 using HLA-VBSeq Software to predict HLA typesBWA version 0.5.9
?. [Structual Variants (SV) Analysis using GASV]
Geometric Analysis of Structural Variants (GASV) Version: 2.0
?. [Gene Integration detection using string search]
Samtools version 1.3
?. [Mycoplasma Contimination detection using BWA]
BWA version 0.5.9Samtools version 1.3.1
TITLE: 2-step PCR mixture and conditions (Barcoded-head primers for seqs pooling)
AUTHORS: Yin-Tse   Huang, Tsu-Chun Hung

[DESCRIPTION]
PCR mixture and condition (2X SUPERGREEN PCR MASTER MIX)

[STEPS]
1. Wear glove, clean up the working bench w. 1% bleach
SECTION: For 1&#x27; PCR head-primers
2. Prepare 1' PCR master mixutre for head-primers (prepare 1.2X of solutions for pipetting error if needed)
 
PCR mixture for head-primers for each reaction
 

 

SECTION: For 1&#x27; PCR head-primers
3. Mix the 1' PCR master mixture gently by pippeting. Quick spin the tube.
SECTION: For 1&#x27; PCR head-primers
4. Transfer  15 µL 1' PCR master mixutre in 8-strip PCR tubes.

SECTION: For 1&#x27; PCR head-primers
5. Add 0.6 µLDNA template in 8-strip PCR tubes, resulting in a 15.6 µL reaction mixture for 1' PCR.


 

SECTION: For 1&#x27; PCR head-primers
6. Mix the reaction mixture gently by tapping the tubes. Quick spin the tubes.
SECTION: For 1&#x27; PCR head-primers
7. Carry out PCR using the following condition:  

1' PCR condition for head-primers
 

SECTION: For 1&#x27; PCR head-primers
7.1. 1' hear-primers used in Huang lab
 

SECTION: For 1&#x27; PCR head-primers
8. Carry out electrophoresis for inspection of DNA products

   

SECTION: For 1&#x27; PCR head-primers
9. Markdown wells and upload the pictures to the Lab Google drive

  

SECTION: For 2&#x27; PCR barcoded-head primers
10. Prepare 2' PCR master mixutre for barcoded-primers (prepare 1.2X of solutions for pipetting error if needed)
 
PCR mixture for barcoded-primers for each reaction (NO PRIMERs at this point!!)
 

 

SECTION: For 2&#x27; PCR barcoded-head primers
11. Mix the 2' PCR master mixture gently by pippeting. Quick spin the tube.
SECTION: For 2&#x27; PCR barcoded-head primers
12. Transfer 21.5 µL of the 2' PCR master mixture to 8-strip PCR tubes.

SECTION: For 2&#x27; PCR barcoded-head primers
13. Add 2.5 µL pre-mixed barcoded-head primers (Forward + Reverse) to each PCR tubes.

SECTION: For 2&#x27; PCR barcoded-head primers
14. Add 1 µL of 1' PCR product as template, resulting in 25 µL reaction mixture for 2' PCR.

Negative control contains only 24 µL master mixture and premixed barcoded-head primers but not DNA template
SECTION: For 2&#x27; PCR barcoded-head primers
15. Mix gently by tapping the tubes. Quick spin the tubes.
SECTION: For 2&#x27; PCR barcoded-head primers
16. Carry out 2' PCR using the following condition:  

2' PCR condition for barcoded-head primers
 

SECTION: For 2&#x27; PCR barcoded-head primers
17. Carry out electrophoresis for inspection of DNA products

  

SECTION: For 2&#x27; PCR barcoded-head primers
18. Markdown wells and upload the pictures to the Lab Google drive
TITLE: Purification of  FKBP8-GST
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol describes the purification and analysis of FKBP8-GST by SDS-PAGE and Coomassie staining.

[STEPS]
SECTION: Purification procedure
1. To purify FKBP8-GST, fuse the cytosol-exposed domain of FKBP8 (1-391aa) to a C-terminal GST-tag and clone into a pET-DUET1 vector. 

SECTION: Purification procedure
2. After the transformation of the pET-DUET1 vector encoding FKBP8-GST in E.coli Rosetta pLysS cells (Novagen Cat# 70956-4), grow cells in 2x Tryptone Yeast extract (TY) medium at 37 °C until an OD600 of 0.4 and then continue at 18 °C. 

SECTION: Purification procedure
10. Elute the proteins with SEC buffer.

SEC Buffer:

 
 
SECTION: Purification procedure
8. To collect the supernatant, collect the beads by centrifugation. Wash the beads twice with 4 mL of wash buffer, and collect the supernatant. 
 
SECTION: Purification procedure
7. Incubate the beads 120 min with 4 mL of 50 millimolar (mM) reduced glutathione dissolved in wash buffer at 4 °C, to elute FKBP8-GST from the beads. 

 Wash Buffer:
 
 
 
SECTION: Purification procedure
6. Centrifuge the samples to pellet the beads and remove the unbound lysate. Then wash the beads twice with wash buffer, once with high salt wash buffer, and two more times with wash buffer. 

Wash Buffer:
 
 
 
 Salt wash Buffer:

 
 
SECTION: Purification procedure
5. Sonicate the cell lysates twice for 30 s and clear by centrifugation at 18000 rpm, 45 min, 4 °C in a SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific). 

Collect the supernatant and incubate with pre-equilibrated Glutathione Sepharose 4B beads (GE Healthcare) for 120 min at 4 °C with gentle shaking to bind FKBP8-GST. 

SECTION: Purification procedure
4. Collect the cells by centrifugation and resuspend in lysis buffer.
 
Lysis buffer:

 
 
SECTION: Purification procedure
3. Once the cells reach an OD600 of 0.8, induce the protein expression with 100 micromolar (µM) isopropyl β-D-1-thiogalactopyranoside (IPTG) for 960 min at 18 °C. 

SECTION: Purification procedure
9. Pool the supernatant fractions, filter through a 0.45 µm syringe filter, concentrated with 30 kDa cut-off Amicon filter (Merck Millipore), and load onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva). 

SECTION: Purification procedure
11. Analyze the fractions by SDS-PAGE and Coomassie staining. Pool the fractions containing purified FKBP8-GST. 

SECTION: Purification procedure
12. After concentrating the purified protein, aliquot the protein and snap-frozen in liquid nitrogen. 

SECTION: Purification procedure
13. Store proteins at -80 °C.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The following is a general procedure guide for preparation and staining of formalin-fixed, paraffin-embedded tissues using a purified, unconjugated primary antibody, biotinylated secondary antibody and streptavidin-horseradish peroxidase (Sav-HRP) and DAB detection system. Because each antigen differs in terms of requirement for fixation, amplification step, etc., it is not possible to write an inclusive protocol that will work for all antigens. The user must determine optimal conditions for each antigen of interest. Many protocols for staining individual antigens, as well as useful tips and troubleshooting guides for immunohistochemistry, can be found at the IHC World web site (<a href="http://www.ihcworld.com/" target="_blank">http://www.ihcworld.com/</a>).</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
How to make a 1 M PMSF Stock Solution

[GUIDELINES]
PMSF is hygroscopic and is highly unstable in aqueous solutions. A typical working solution of PMSF is 1mM.<br /><br /><a href="http://store.p212121.com/pmsf/" target="_blank">PMSF</a> is an anti-protease! Do not allow to come into contact with skin.<br /><br />PMSF is inactivated in aqueous solutions. The rate of inactivation increases with increasing pH and is faster at 25&deg;C than at 4&deg;C. The half-life of a 20mM aqueous solution of PMSF is ~35 minutes at pH 8.0.&nbsp;This short half-life means that aqueous solutions of PMSF can be safely discarded after they have been rendered alkaline (pH &gt;8.6) and stored for several hours at room temperature.

[STEPS]
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TITLE:  OCT Embedding
AUTHORS: Joanna Bi

[DESCRIPTION]
OCT Embedding protocol 

[STEPS]
1. Sample tissue is collected fresh.
2. Squeeze a thin layer of OCT compound (Sakura Tissue-Tek O.C.T. Compound, ref 4583) into a disposable mold.
3. Place the sample tissue in the center of the mold, with the side to be cut oriented into the mold. 
4. Squeeze more OCT compound over the top to full cover the tissue.
5. Lower the mold into liquid nitrogen, being careful to not fully submerge the mold. Hold in liquid nitrogen for a few seconds until fully frozen.
6. Place frozen mold in dry ice until stored in -80ºC
TITLE: BSCI:414--Lab 13 Western Blot of Spike and Spike(RBD)
AUTHORS: Harley King

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">We used an anti-histidine polyclonal rabbit antibody as a primary antibody. The secondary antibody was a goat anti-rabbit antibody with conjugated HRP. </div></div>

[STEPS]
?. Watch this video to understand the WB process.
?. Download the WB and Coomassie images and save them to your lab notebook.
?. Read about the primary antibody.Read about the secondary antibody.
TITLE: PCR protocol for gene COXI neo-caledonian freshwater micro-crustaceans
AUTHORS: coline.royaux, Nicolas Rabet, Céline Bonillo

[DESCRIPTION]
This PCR protocol has been used to amplify the Cytochrome Oxydase I gene of neo-caledonian genera Boeckella, Lynceus, Eulimnadia and Streptocephalus with several primers.

[STEPS]
1. Prepare the mix for the PCR depending on the quantity of DNA samples you want to amplify
 

 



1.1. For each well, mix 12.44 µL, 2 µL, 1 µL, 1 µL, 0.8 µL and 0.12 µL. This mix is hereafter named "intermediary mix".
 


1.2. Add your primers to the mix, 0.32 µL 10 picomolar (pM) and 0.32 µL 10 picomolar (pM)  for each well. This mix is hereafter named "final reagent mix".
 

 



2. In each well, deposit 18 µL and 2 µL unless for the negative control.
For the positive control, use the DNA of the species on which the primer has been designed.

3. Close the PCR plate with a heated aluminium foil and centrifuge it enough to make sure the reagents are all in the bottom of the well.
4. To amplify targeted DNA sequences, place your plate in the thermocycler, set and launch the cycle as described in the following sub-steps
4.1. 5 min at 94 °C 

4.2. 40 cycles of the following steps :
30 s at 94 °C 
30 s at 50 °C 
1 min at 72 °C 

4.3. 8 min at 72 °C 

4.4. Infinite timing at 20 °C so the samples are well preserved and you have some time to pick it up

5. To know if your amplifications worked properly, you now have to proceed to a gel electrophoresis of your PCR products
5.1. To prepare the gel,
 

 mix 0.8 mg and 40 mL, heat the preparation in the microwave for a few moments until it is homogeneous (you can agitate the preparation). Wait until the preparation is cold enough to touch its container and add 0.8 µL.
5.2. Pour the preparation in a mold and let the gel polymerize with a bar to form wells to deposit your PCR products
5.3. Deposit the gel in the electrophoresis tank
5.4. Fill each well with 2.5 µL and 0.5 µL , don't forget to save a well to deposit 1.2 µL 

5.5. Start electrophoresis in TAE buffer for 15 min at 200 Volts

5.6. Look at your gel under Ultra Violet light to see the results
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This is the protocol accompanying the "pLKO.1 &ndash; TRC Cloning Vector". For information about the PLKO.1-TRC cloning vector and tips on designing shRNA oligos for pLKO.1 see Addgene's website: http://www.addgene.org/tools/protocols/plko/

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>1)The partial E gene of dengue virus is amplified before sequencing by using four sets of serotype-specific <br />   oligonucleotides as referenced in the manuscript text.</p>
<p>2) The reaction mixture is prepared as the following:</p>
<p>    12.5ul of MyTaq RT-PCR (Bioline, Korea)</p>
<p>      4.5ul of  Nuclease-Free Water</p>
<p>      1.0 ul of Foward Primer for the respective serotype  (10uM)</p>
<p>      1.0 ul of Reverse Primer for the respective serotype (10uM)</p>
<p>      0.5ul of RNase inhibitor</p>
<p>      0.5ul of Reverse Transcriptase (RT)</p>
<p> 3) Aliquot 20ul in each PCR tubes.</p>
<p> 4) 5ul of Dengue RNA (with known serotype) is added to the corresponding PCR tube.</p>
<p> 5) PCR amplification is performed on CFX-96 (BioRad) conventional PCR thermal cycler.</p>
<p> 6) The PCR is performed with the following cycling profile: 35 cycles (94 °C for 1 min, 55 °C for 1 min and 72 °C for 1.5 min) followed by an extension reaction at        72 °C for 5 min.</p>
<p> 7) A 25 µl aliquot of each PCR reaction is analyzed on 1.5 % pre-stained agarose by gel electrophoresis, run for 30 min at 90 V.</p>
<p> 8) The gel is then viewed under UV illumination.</p>
<p> 9) The expected amplicon size for each serotype is 578bp for DENV1, 617bp for DENV 2, 582bp for DENV3 and 572bp for DENV 4 The corresponding <br />     amplicons are extracted from the agarose gel and purified by Gel Extraction Kit (Qiagen, USA) according to the manufacturer ’s instruction.</p>
<p> 10) The final elution volume is 30 µl of purified PCR amplicons. Then, 5 µl of these are reanalyzed on 1.5 % agarose gel to substantiate the accuracy of <br />      purification step. The purified PCR amplicons were outsourced for sequencing.</p>

[STEPS]
TITLE: MojoSort™ Human CD45 Nanobeads Protocols
AUTHORS: Sam Li

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Note: If the percentage of CD45+ cells in your sample is less than 50%, please follow Protocol 1. If it is higher than 50% then please follow protocol 2.</span></div><div class = "text-block">The cells targeted by the Nanobeads are either selected or depleted by incubating your sample with the directly conjugated magnetic particles. The magnetically labeled fraction is retained by the use of a magnetic separator. After collection of the targeted cells, downstream applications include functional assays, gene expression, phenotypic characterization, etc.</div></div>

[STEPS]
TITLE: Operant Conditioning
AUTHORS: daniel.dautan daniel, Per Svenningsson

[DESCRIPTION]
This protocol is for training mice operant conditioning to assess cognitive performance using the resources described in Matikainen-Ankney BA et al 2021.

[STEPS]
SECTION: &quot;Follow the light&quot; testing
9. The number of correct, incorrect pokes and pellets collected within 10s
of delivery were then extracted for further analyses.
SECTION: &quot;Follow the light&quot; testing
8. Finally, on the last day, switch the protocol to "Follow the Light" for 24 hours. 
Associate the poke  with pellet delivery with a yellow LED light turning ON in the nose poke. 
When a poke occurrs on the correct side, deliver a 20mg sugar pellet with an auditory cue
Pair incorrect poke with a visual cue. 
Similar to the previous protocols, when a pellet is delivered, inactivate both correct and incorrect nose pokes to prevent multiple pellet deliveries. 
7. After three consecutive sessions of FR1, increase the protocol to Fixed Ratio 3 (FR3) for one day. 
Each time the mouse produces three correct nose pokes, deliver a 20mg pellet with the same auditory cue. 
Pair incorrect pokes with a visual cue to indicate errors. 
Similar to FR1, when a pellet is delivered, both correct and incorrect nose pokes are inactivated to avoid multiple pellet deliveries. 
6. For the next 3 days, switch to a Fixed Ratio 1 (FR1) protocol starting at 09:00. 
Pair each correct poke with an auditory cue and the delivery of a single sugar pellet, while pairing each incorrect poke was a visual cue (Blue LED positioned below the pellet dispenser). 
When a pellet is delivered, program so that both correct and incorrect nose pokes became inactive
to prevent multiple pellet deliveries. 
5. For the following two days, expose each mouse to free-feeding sessions where 20mg sugar pellets (BioServ) are randomly delivered to habituate the mice. 
SECTION: Operant box set-up
3. Define the FED 3.0 operant box with two nose pokes using infrared beams to determine nose entry, and position a food pellet dispenser in the middle.
SECTION: Operant box set-up
2. In each animal cage, position a FED3.0 operant box. To prevent wood bedding from entering the nose poke, provide reduced bedding amounts to each animal. 



SECTION: Operant box set-up
1. Singly house and food restrict the mice.
SECTION: Pellet training
4. Train mice to the FED3.0 for 24 hours with 6g of chow pellets placed on the floor. 
SECTION: Analysis
10. Extract the following measures:
The number of correct pokes
The incorrect pokes 
Pellets collected within 10s of delivery
TITLE: Preparation of DEPC-treated water
AUTHORS: Carlos Costas Insua

[DESCRIPTION]
Diethyl pyrocarbonate (DEPC) is a wide-spectrum RNase inhibitor. DEPC-treated water is generally used when working with RNA

[STEPS]
1. Add 1 mL of fresh DEPC to 1 L of H2O. Shake well to disperse the DEPC through the H2O. Incubate at 37°C for at least 12 h and/or autoclave at 15 psi on liquid cycle for 20 min to inactivate the remaining DEPC.