eamag/protocols-with-steps huggingface dataset:
TITLE: Primary Lung Fibroblasts (PLF) Plating/Freezing Protocol
AUTHORS: Morrisey Lab

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Primary Lung Fibroblasts (PLF) Plating/Freezing Protocol</span></div><div class = "text-block"><span style = "font-weight:bold;">PLF Media </span></div><div class = "text-block">90 mL DMEM NUTRIENT MIX F12 (Invitrogen/Cell Center, cat# 11320033)</div><div class = "text-block">10 mL 10% FBS (Invitrogen, cat#10437-028)</div><div class = "text-block">1 mL ANTIBIOTIC ANTIMYCOTIC-Anti-Anti 100x ( Invitrogen/Cell Center, cat#15240096)</div><div class = "text-block"><span style = "font-weight:bold;">Freeze Media </span></div><div class = "text-block">40 mL PLF Media</div><div class = "text-block">5 mL 10% FBS (Invitrogen, cat# 10437-028)</div><div class = "text-block">5 mL DMSO (Sigma, cat#D2650-100ml)</div><div class = "text-block">Nunc™ EasYFlask™ Cell Culture Flasks, T75, filter (hermo/ Life Tech, cat#156499)</div><div class = "text-block">TRYPSIN 0.05% EDTA (Invitrogen/Cell Center, cat#25300054)</div><div class = "text-block">CryoVials (Neta Sci, cat#430659)</div></div>

[STEPS]
TITLE: Soil sample(Citizen scientists, Chinese)
AUTHORS: Hsin-Mao   Wu

[DESCRIPTION]
Soil sample, citizen scientist

[STEPS]
SECTION: 採土
1. 選定地點，分為以下三種
醫院
公園
天然地區

SECTION: 採土
2. 移除表土，取距離土表5~7公分之處的土，裝入塑膠夾鏈袋中
SECTION: 採土
3. 將塑膠袋中的土均勻混合，分別倒入兩個HDPE塑膠罐之中
 

SECTION: 採土
4. 將HDPE罐子收進袋子中
SECTION: 採土
5. 最後拍兩張照片
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<span data-reactid=".0.1.0.1.0.0.0.4.0">Quick and easy method to transform a free plasmid into either budding or fission yeast.</span>

[BEFORE_START]
<span data-reactid=".0.1.0.1.0.0.2.1.1.0">Grow at least a 1 mL liquid yeast culture overnight, or plate yeast on minimal medium overnight for fresh yeast.</span>

[STEPS]
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?.
TITLE: Determining Protein Concentration of Cell-free Extract
AUTHORS: Weston Kightlinger

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Cell-free protein synthesis reactions require high-quality, active extracts to produce high yields of protein therapeutics, enzymes, antigens, vaccines, and other proteins. One way of assessing extract quality and lysis efficiency during cell-free extract preparation is to determine its overall protein concentration. This can be done using a Bradford assay.</div><div class = "text-block"><span>Read more on cell-free protein synthesis here: </span><a href="https://www.swiftscalebio.com/blog/cell-free-protein-synthesis" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.swiftscalebio.com/blog/cell-free-protein-synthesis</span></a><span style = ":;"> </span></div></div>

[STEPS]
?. [Determining Protein Concentration of Cell-free Extract]
In 96 well plate with lid 3370 add  of Bradford Reagent to each well.
250 µl
?. [Determining Protein Concentration of Cell-free Extract]
Dilute BSA standards according to the following:
?. [Determining Protein Concentration of Cell-free Extract]
Make a serial dilution of extract (expected concentration of cell-free extract is ~).
?. [Determining Protein Concentration of Cell-free Extract]
Measure all samples in triplicate [(# of samples+ 8 standards)*3=# of wells used].
?. [Determining Protein Concentration of Cell-free Extract]
Add  of sample/standard to each well containing Bradford Reagent.
5 µl
?. [Determining Protein Concentration of Cell-free Extract]
Place plate on plate reader (must be able to read absorbance at 595 nm).
?. [Determining Protein Concentration of Cell-free Extract]
a. Turn on lamp and allow to warm up.
?. [Determining Protein Concentration of Cell-free Extract]
b. Set which wells to read in both protocol and procedure.
?. [Determining Protein Concentration of Cell-free Extract]
c. Plot standard curve using preferred software and use to calculate protein concentration from sample signal and dilution factor.
Expected Result: Expected concentration of protein in Cell-free extract is ~  so make the following dilutions:
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.

Negative control contains only 15 µL master mixture but not DNA template

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: UC Davis - Mouse Model Creation
AUTHORS: Kent Lloyd

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">This is a generic protocol for making genetically-altered mouse models for research. Investigators who do not have a mutant mouse to send for testing at the MMPC @ UC Davis can ask for the mutant mouse to be derived by the MMPC @ UC Davis, through the UC Davis Mouse Biology Program. Genetically altered mice can be made on most genetic backgrounds, such as C57BL/6, FVB/N, B6D2F1 and other hybrids, and others. Transgenic (random genomic integration of expression-competent transgenes) and gene targeted (specific insertion and/or replacement of an endogenous genetic locus) mutant mice can generated.  “Knockout” mice can be made so as to delete endogenous gene expression ubiquitously and conditionally in either a specific cell or tissue type (spatial conditional) and/or at a specific age (temporal conditional). Numerous technologies are available for the derivation of mutant mice, including molecular construct design, gene targeted in embryonic stem (ES) cells, pronuclear microinjection of transgenic constructs into zygotes, ES cell microinjection into blastocysts, genome editing in ES cells and zygotes (e.g., TALENs, CRISPR/Cas9).  Mutant mice can be genotype confirmed (see MMPC item D2004) and custom bred to generate cohorts of male and/or female mice for phenotyping at the MMPC @ UC Davis. </div></div>

[STEPS]
?. Depending on the type of mutant mouse requested, a number of protocols can be used.  Investigators should consult with technical assistance staff at the MMPC @ UC Davis to develop their plan for deriving a mutant mouse.
TITLE: Creation of HBSS Wash Buffer | Brusko Lab
AUTHORS: Brusko Laboratory, Matthew Brown, Thinzar Pe Myint

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

[STEPS]
?. Open a sterile 1 liter (L) Rapid-Flow Sterile Disposable Filter Unit with a 0.2 micrometer (µm) PES Membrane.
?. Remove the lid of the filter flask and add   of (1x) HBSS to the filter.
940 ml
?. Add a single   aliquot of FBS and a  aliquot of Penicillin/Streptomycin (Pen/Strep) to the HBSS in the filter system
50 ml
10 ml
?. Add  of   EDTA to the solution.
10 µl
?. Process the final solution through the 0.2 µm PES membrane  using a positive-pressure system.
?. Once the solution has been filtered, remove the filter from the flask by unscrewing the filter and dispose of the filter into biohazardous waste.
Since this procedure makes use of penicillin and streptomycin, any disposable products that come into contact with these antibiotics should be disposed of in biohazards waste to prevent the creation of antibiotic-resistant bacteria.
?. Remove the flask lid from the secondary container and securely screw the cap onto the flask. Label the flask containing the sterile HBSS Wash Buffer with the following information: "HBSS with FBS, EDTA, Pen/Strep" as well as your name and the date.
?. Store the HBSS Wash Buffer in   .
4 °C
TITLE: The annotation pipeline for the genome of a snake
AUTHORS: Boyang Liu, Liangyu Cui, Zhangwen Deng, Yue Ma, Diancheng Yang, Yanan Gong, Yanchun Xu, Shuhui Yang, Song Huang

[DESCRIPTION]
Here are detailed methods use for the annotation of various snake genomes. 

[STEPS]
SECTION: Repeat annotation_de novo
1. 1) Run RepeatModeler to build a de novo library based on the input assembled genome sequence.
2) Using the library constructed in step 5 as the database, run RepeatMasker (v. 3.3.0) to find and then classify the repetitive sequences.
 
SECTION: Repeat annotation_database
2. Run TRF (v. 4.09), RepeatMasker and RepeatProteinMask (v. 3.3.0) to identify repeats in the genome at DNA and protein level, respectively, by aligning sequences against the Repbase library (v. 17.01).
 
SECTION: Gene prediction_preparation
3. Mask these repetitive regions obtained above (step 4-6)  with 'N's.
 
SECTION: Gene prediction_de novo
4. Run Augustus (v3.0.3)  to de novo predict genes in the repeat-masked genome sequences.
 
SECTION: Gene prediction_homolog
5. Download the publicly available protein sequences of representative homologous snake species, align these against our masked genome sequences with BLAT, and then based on the BLAT mapping results, GeneWise (v2.4.1 ) is then run to predict the genes.
 
SECTION: Gene prediction_transcriptome
6. Then filter RNA-seq data using Trimmomatic(v0.30). The resulting data is then assembled by Trinity (v2.13.2). PASA(v2.0.2) was finally used to align transcript against the snake genome of interest to obtain gene structures.
 
SECTION: Final gene set_MAKER
7. Integrate the genes predicted in step 4-6 to obtain the consensus gene set using the MAKER pipeline (v3.01.03).

SECTION: Functional annotation
8. Map protein sequences of the final gene set to existing databases to identify their functions or motifs, such as SwissProt, TrEMBL, KEGG, InterPro.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol for isolating environmental DNA from filter membranes is modified from Renshaw et al. 2015.</p>
<p>The room temperature preservation of filtered environmental DNA samples and assimilation into a phenol–chloroform–isoamyl alcohol DNA extraction.</p>
<p>Molecular Ecology Resources 15(1): 168-176.</p>

[STEPS]
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TITLE: Western Blot (tank-blot) + Antibody staining
AUTHORS: iGEM Dusseldorf

[STEPS]
?. [Western blot]
Create blotting buffer stock containing:100mL 10x SDS running buffer 200mL 100% Methanol700mL H20Fill a tray with blotting buffer
?. [Western blot]
Transfer SDS gel to the blotting buffer
?. [Western blot]
Assemble blotting sandwich (take it, soak sponge and place on it, soak filter paper and place on it, place inverted SDS gel on top without creating bubbles)
?. [Western blot]
Add charged PDVF membrane with forceps (charged in 100 % ethanol and than soaked in buffer)
?. [Western blot]
Add soaked Filter and Streak out bubbles
?. [Western blot]
Add soaked sponge
?. [Western blot]
Press togehter and close container
?. [Western blot]
Place in blotting apparatus and add blotting buffer
?. [Western blot]
Run for 1 h at 100 V 350-500 mA
?. [Antibody staining ]
Add 40 ml resh TBS-BSA in empty pipet tip box.
?. [Antibody staining ]
Place membrane from blottig appartus with forceps into the solution
?. [Antibody staining ]
Protein side needs to be up
?. [Antibody staining ]
Incubate overnight at 4° C with shaking 20 rpm
?. [Antibody staining ]
Discard blocking solution
?. [Antibody staining ]
50 ml 1x TBS-BSA, 100 µl Tween-20 and x µl Antibody (dependend on the affinity)
?. [Antibody staining ]
Incubate for 1 h with shaking
?. [Antibody staining ]
Wash 2 times with TBST quickly , then wash 3 times a 10 min with TBST
?. [Antibody staining ]
Develop using ECL ( enhanced chemiluminescence) with HRP
?. If you are work with a secondary antibody icubate in the secondary antibody solution and repeat steps 17
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol outlines the preparation of a basic sucrose loading dye 10 X stock solution for gel electrophoresis (DNA/RNA). Orange G runs at approximately 50 bp making this loading dye ideal for small molecular weight polynucleotides. Used mainly as an indicator for approx. 100 bp polynucleotides, where we would like to avoid contamination of small fragments with other loading dyes. It is also useful as an indicator if you have excessive primer-dimer amplification in your PCRs as these usually also run at approx. 50 bp.</p>
<p> </p>
<p>The origins of this protocol have been lost in the mists of time and this method been handed down from PhD student to PhD student.</p>
<p> </p>
<p> </p>

[STEPS]
?. 
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?. 
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?. 
?.
TITLE: Tissue staining for IMC
AUTHORS: Santhosh  Sivajothi

[DESCRIPTION]
FFPE tissue processing for IMC

[STEPS]
1. Warm slide to 60C for 10 mins on a slide heater
2. Dewax the slide in Histoclear for 20 min in the fume hood.
3. Rehydrate the slide in descending grades of ethanol (100%, 95%, 90%, 70%, 50%), 5 min each.
4. During rehydration, prepare a coplin jar with 1X antigen retrieval solution and place in pressure cooker to warm.
5. Rinse the slide in distilled, deionized water for 5 min.
6. Insert the slide with tissues into the pre-heated antigen retrieval solution. Perform antigen retrieval under low pressure setting, heating the solution to 95C for 15 mins.
7. Following incubation, release the pressure and place the jar containing the antigen retrieval solution and slides on a lab bench and cool to room temperature, approximately 30 min (or until it reaches room temperature).
8. Rinse the slide with distilled, deionized water for 7 min.
9. Wash the slide with DPBS (no Ca or Mg) for 7 min.
10. Use a PAP pen to encircle the tissue.
11. Block the tissue with 3% BSA in DPBS (no Ca or Mg) or commercial blocking solution such as SuperBlock for 45 min at room temperature. Use enough block solution to cover the tissue, normally around 300 μl/section.
12. Determine antibody working concentrations by prior experiments using a series dilution of identical antibody conjugates.
13. Prepare antibody cocktails. Microcentrifuge metal-conjugated antibodies individually at maximum speed for 3 min prior to making the antibody cocktail, and add only the supernatant. Calculate the total volume of antibodies needed at concentrations specific for the assay (dilute as necessary using 0.5% BSA in DPBS).
14. Incubate tissue sections with antibody cocktail overnight at 4C in a hydration chamber. Antibody cocktail volume should be sufficient to cover the tissue, about 100 μl.
15. Following overnight incubation, wash the slide twice in 0.1% Triton-X in DPBS (no Ca or Mg), each time for 7 min.
16. Wash the slide twice in DPBS (no Ca or Mg), each time for 7 min.
17. Stain the tissue with intercalator-Ir (1:500 dilution) in DPBS (Ca orMg) for 5 min at room temperature.
18. Rinse the slide in distilled, deionized H2O for 1 min.
19. Air-dry the slide under a gentle flow of pressurized air to dry quickly. After drying completely, proceed with acquisition on Hyperion instrument.
TITLE: Stereo-seq cell type abundance analysis
AUTHORS: Peter Kilfeather

[DESCRIPTION]
Stereo-seq cell type abundance analysis from Kilfeather, Khoo et al., 2024

[STEPS]
SECTION: Protocol
1. To test for differential abundance of cell types between age groups, we used mixed effects modelling of associations of single cells (MASC). MASC tests whether cell type membership of individual cells is influenced by an experimental covariate of interest, while accounting for technical covariates and biological variation. We specified mouse genotype as a fixed covariate and mouse of origin as a random effect in the generalized, mixed-effect model. Changes in cell type abundance were considered significant at FDR-P < 0.01.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes how Aiptasia stocks are cared for in the Weis lab. The protocol was last updated in 2018. Most students and postdocs tha have been in the Weis lab have contributed to this protocol in some form or another, and they are too numerous to list.</p>

[BEFORE_START]
<p>For basic Aiptasia care, the ability to control lighting and temperature are necessary. Aiptasia are hearty creatures, and can tolerate living in a normal fish aquarium or in small plastic food dishes. Just like with other marine life, good water quality is crucial for survival.</p>

[STEPS]
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TITLE: BFM speed recording with back-focal-plane interferometry
AUTHORS: Teuta Pilizota, Ekaterina Krasnopeeva, Jerko Rosko

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The speed of bacterial flagellar motor (BFM) is measured with back-focal-plane interferometry. Heavily attenuated optical trap (855 nm laser) is used to detect the rotation of a polystyrene bead attached to a truncated flagellar filament. Time course of the bead rotation is recorded with the position-sensitive detector.</div></div>

[STEPS]
?. [Preparing the cells]
Grow cells to the desired OD
?. [Preparing the cells]
Truncate flagellar filaments by passing a bacterial suspension through two syringes with narrow-gauge needles (26 gauge) connected with a plastic tube (“shearing device”) 30-80 times
?. [Preparing the slide]
Coat the surface of the tunnel-slide (or flow-cell) with 0.1% poly-L-lysin (PLL) by flushing PLL through the flow-cell/tunnel-slide for 10-20 s followed by washing it out with the excessive volume of the experimental medium (~20 times the volume of the channel).
?. [Preparing the cells]
Wash cells into the experimental medium 3 times by centrifugation at 8000*g for 2 minutes. We use microcentrifuge tubes to spin the cells down and resuspend the pellet in 1ml of the experimental medium. At the end of the wash resuspend the cells in the experimental medium to the desired concentration.
?. [Preparing the slide]
Load sheared and washed cells into the flow-cell/tunnel-slide and incubate for 10 min to allow attachment.  Wash excessive cells out with the experimental medium.
?. [Preparing the slide]
Add 0.5 μm in diameter polystyrene beads (Polysciences, Inc, USA) resuspended in the experimental medium to the flow-cell/tunnel-slide and incubate for 10 min with consequent washing out of the non-attached beads.
?. [Microscopy]
Place a potential spinner into the focus of 855 nm laser.
?. [Microscopy]
Find the best position to see a circular trajectory and record the speed with 10 kHz sampling rate with the position sensitive detector with a 2.5 kHz cutoff anti-aliasing filter applied.
TITLE: GG2 - CRISPR-Cas9 episome cloning using red-blue screening for Phaeodactylum tricornutum
AUTHORS: Mark Moosburner, Andrew Allen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">To ensure Cas9 activity upon episomal transformation, the 3’ end of the Cas9 coding sequencing was transcriptionally fused to an antibiotic selectable marker, shble, by the 2A peptide. The Cas9-2A-shble construct was cloned into a Phaeodactylum episome that included a bacterial expression cassette for a red fluorescent protein (RFP). The RFP was also flanked by two BsaI restriction digest sites. The RFP vector with Cas9 was used as a cloning vector to assembly one or more sgRNA Phaeodactylum expression cassettes and a single LacZ bacterial expression cassette in place of the RFP. Upon successful assembly, the correct colonies appear blue on selective agar plates while incorrect colonies appear either red or colorless. The golden gate assembly protocol used to synthesize the episome (g51092-2, g24739-A) is as follows:</div></div>

[STEPS]