eamag/protocols-with-steps huggingface dataset:
TITLE: Immunostaining
AUTHORS: Cole S Sitron, Victoria A Trinkaus, F Ulrich Hartl

[DESCRIPTION]
This is a protocol that describes how to use antibody staining to detect cellular epitopes by immunofluorescence microscopy.


[STEPS]
SECTION: Fixation
1. Aspirate media and gently wash each well with 1X PBS.

SECTION: Fixation
2. Aspirate PBS and place 500 µL of 4% paraformaldehyde onto each well.

SECTION: Fixation
3. Fix at Room temperature for 10 min.

SECTION: Fixation
4. Remove 4% paraformaldehyde and add 1 mL 1X PBS.

 

SECTION: Immunostaining
5. Permeabilize 1 mL 0.1% Triton X-100 and gently shake at Room temperature for 5 min.

SECTION: Immunostaining
6. Block in 1 mL Blocking Buffer for 60 min.

SECTION: Immunostaining
7. Dilute primary antibodies into Blocking Buffer and add 300 µL of diluted antibodies to each well.

SECTION: Immunostaining
8. Shake at 4 °C 60 min.

SECTION: Immunostaining
9. Wash 3x with 1 mL PBS, gently shaking at Room temperature each time for 5 min.

SECTION: Immunostaining
10. During washes, centrifuge tubes of secondary antibodies at 16000 x g for 10 min at 4 °C.

SECTION: Immunostaining
11. Dilute secondary antibodies into Blocking Buffer 1:500.

SECTION: Immunostaining
12. Incubate coverslips in 300 µL diluted secondary antibody for 2-3 hr at Room temperature with gentle shaking, protected from light.

SECTION: Immunostaining
13. Wash with 1 mL PBS for 5 min with gentle shaking.

SECTION: Immunostaining
14. Add 1 mL diluted NucBlue and incubate with gentle shaking for 5 min, protected from light.

SECTION: Immunostaining
15. Wash 2x with 1 mL PBS for 5 min with gentle shaking.

SECTION: Immunostaining
16. Allow fluorescence mounting medium to come to Room temperature.

SECTION: Immunostaining
17. Add one drop of fluorescence mounting medium onto microscope slides for each coverslip.
SECTION: Immunostaining
18. Mount coverslips onto slides.
SECTION: Immunostaining
19. Let slides cure at Room temperature 5 min, protected from light.
TITLE: PCR (Error-prone PCR)
AUTHORS: Zhujun Wei

[STEPS]
?. Set up a small box with ice, put the tubes of DNA, 2 x Mut Random System, Mut Enhancer and ddH2O into it before loading them into the Bio-rad S1000TM Thermo Cycler.
?. Add the following reagents to a PCR tube (20 μL).  AB1IngredientVolume2Template Plasmid(1-10 ng/µL)0.4 µL32 x Mut Random System10 µL4Forward Primer (10 μM)0.4 µL5Reverse Primer (10 μM)0.4 µL6Mut Enhancer0-20 µL7ddH2OTo 20 µL
AB1IngredientVolume2Template Plasmid(1-10 ng/µL)0.4 µL32 x Mut Random System10 µL4Forward Primer (10 μM)0.4 µL5Reverse Primer (10 μM)0.4 µL6Mut Enhancer0-20 µL7ddH2OTo 20 µL
?. Program the thermocycler as follows:  AB1Temperature Time295℃2 min394℃30 s455℃1 min572℃1 min/kilo basepairs672℃7 min74℃∞Note: Higher initial template concentrationscan lead to lower mutation rates. More amplification cycles may cause higher mutation rates.
AB1Temperature Time295℃2 min394℃30 s455℃1 min572℃1 min/kilo basepairs672℃7 min74℃∞
?. Build a mutant library, expand the strains, and add the corresponding inducers, collect data using a Microplate reader operating procedure V.1, and use the GraphPad Prism for data processing and analysis.
?. Select the positive results for DNA sequencing to determine the mutated nucleotides in the DNA promoter or coding sequence.
TITLE: API Synthesis
AUTHORS: BOC      Sciences

[DESCRIPTION]
BOC Sciences’ priority is to provide the highest quality APIs to our customers. Our experts have a wealth of experience in the synthesis of APIs with various starting materials and intermediates. The products will be provided with integrated data packages including chemical purity (GC/MS, HPLC, LC/MS) and concentration (mass spectrometry and NMR). We are pleased to hear from you and looking forward to working with you.

[STEPS]
TITLE: Real time qPCR (Fly Heads)
AUTHORS: Mel Feany

[DESCRIPTION]
This Protocol describes how to perform RT qPCR with RNA extracted from fly heads.

[STEPS]
SECTION: RNA Extraction
1. Homogenize 6 heads in 50 µL Qiazol, then add 450 µL Qiazol.

SECTION: RNA Extraction
2. Spin 10 min  at 4 °C at 12,000 x g.

SECTION: RNA Extraction
3. Incubate supernatant for 5 min at Room temperature .

SECTION: RNA Extraction
4. Add 100 µL  chloroform.  Vortex, incubate for 3 min  at Room temperature , then spin for 15 min  at 4 °C at 12,000 x g.

SECTION: RNA Extraction
5. Transfer the upper phase to fresh tube, add 250 µL isopropanol, incubate 10 min at Room temperature , spin for 10 min  at 4 °C  at 12,000 x g.

SECTION: RNA Extraction
6. Wash the pellet in 500 µL 75% ethanol (it will be very loose) three times.  Do not vortex.

SECTION: RNA Extraction
7. Air dry
SECTION: RNA Extraction
8. Resuspend in 15 µL RNAse/DNAse-free water.

SECTION: RNA Extraction
9. Measure 260/280 and 260/230 on nanodrop.
SECTION: RT-PCR
10. Use 1 µg RNA to perform RT-PCR reaction.

SECTION: RT-PCR
11. Thermocycler settings
a.     25 °C  10 min
b.     37 °C  120 min 
c.      85 °C  5 min 
d.      4 °C Hold

SECTION: qPCR
12. Make master mix with primers, SYBR Green and water.
SECTION: qPCR
13. Dilute DNA 1:50 making a 50 µL stock.

SECTION: qPCR
14. Add 2 µL  DNA to wells with special tips.

SECTION: qPCR
15. Add 14 µL master mix to wells with regular tips. Pipette up and down once but do not expel a bubble. Keep master mix on ice the entire time to avoid bubbles.

SECTION: qPCR
16. Check for bubbles and run on machine.
TITLE: Installation instructions for BEAST and BEAGLE on Avell G1550 RTX MUV/A65 RTX MUV
AUTHORS: Laise Moraes

[STEPS]
?. [Installing dependency packages]
Open a terminal (Ctrl + Alt + T).Update the package list and install the dependency packages for the BEAST and BEAGLE.
?. [Installing Intel OpenCL implementations]
Create a directory called softwares to your HOME directory, switch to it and then download the Intel OpenCL implementations installer.Unpack the compressed .tar.gz file, switch to installer directory and install the Intel OpenCL implementations, accepting defaults.After installation, remove the Intel OpenCL implementations installer directory and installer.
?. [Installing NVIDIA CUDA drivers]
Get the PPA repository and install the NVIDIA CUDA driver.
?. [Installing BEAGLE]
Build the BEAGLE from the source repository.
?. [Installing BEAST]
Download the BEAST installer and unpack the compressed .tgz file.After install, remove the compressed .tgz file.
TITLE: DNA Isolation 
AUTHORS: NUS iGEM

[DESCRIPTION]
2023 NUS-Singapore iGEM team followed this protocol to isolate the DNA fragments from the agarose gel after the gel electrophoresis. 

[STEPS]
1. Prepare and label an Eppondoft tube.
2. Place the agarose gel onto the LED Transilluminator (blue light) to observe the DNA band(s).
 

3. Cut out the target DNA band from the agarose gel.
4. Put the gel piece into the Eppendorf tube.
5. Add 450 µL of   into the Eppendorf tube.

6. Heat the Eppendorf tube at 55 °C for 20 min in the Thermo-Shaker.

7. Add 150 µL of 100% isopropanol (IPA) into the Eppendorf tube and shake the tube to mix the solution well.

8. Transfer the whole solution into a QIAquick Spin Column (purple tube with a maximum volume of 750 µL). 

9. Centrifuge it for 13 rpm, 1 min.

10. Discard the flow-through and place the QIAquick column back into the same tube.
11. Add 700 µL of   into the QIAquick column.

17. Centrifuge the emptied QIAquick column at 13 rpm, 1 min to remove residual  .

18. Transfer the QIAquick column into the newly labelled Eppendorf tube.
19. Add 30 µL of DI water into the QIAquick column.

20. Centrifuge the tube at 13 rpm, 1 min, ensuring that the direction of the Eppendorf tube’s cap is the same as the direction of spinning to avoid breaking.

21. Discard the QIAquick column, the solution left in the Eppendorf tube contains the DNA fragment of interest.
22. Use the Nanodrop to measure and record the purity and concentration of the DNA fragment.
 

12. Centrifuge it for 13 rpm, 1 min.

13. Discard the flow-through and place the QIAquick column back into the same tube.
14. Add 700 µL of   again into the QIAquick column.

15. Centrifuge it for 13 rpm, 1 min.

16. Discard the flow-through and place the QIAquick column back into the same tube.
23. Keep the isolated DNA fragment in the Room temperature.
TITLE: In vitro assembling of RNP for nucleofection of hPSCs
AUTHORS: Hanqin  Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner

[DESCRIPTION]
This protocol describes the procedure for the in vitro assembly of ribonucleoprotein (RNP) which can be delivered into human pluripotent stem cells (hPSCs) using nucleofection.

General notes
1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.





[STEPS]
1. Thaw Cas9 or PE2 protein on ice . We don’t usually reuse leftover protein, since freezing and thawing compromises protein activity.

2. In each nucleofection reaction, prepare 10 µl RNP
3. For regular CRISPR/Cas9 editing, use:
    5 µl, autoclaved H2O
    2 µl, 40 µM purified Cas9 protein
    3 µl, 100 µM synthetic sgRNA
4. For prime editing PE2 strategy, use:
    7-x µl, autoclaved H2O
    x µl (90 pmol), purified PE2 protein
    3 µl, 100 µM synthetic pegRNA
5. For prime editing PE3 strategy, use:
    7-x µl, autoclaved H2O
    x µl (90 pmol), purified PE2 protein
    2 µl, 100 µM synthetic pegRNA
    1 µl, 100 µM synthetic ngRNA
6. Pipet the proper amount of each component in the order showing above into a microcentrifuge tube. Mix using a P10/20 tip. 
7. Incubate at Room temperature  for 10 min  to assemble RNPs. Once assembled, RNPs are stable at room temperature for 30 min, or at least 2h on ice.  

8. If HDR template is desired, mix 100 pmol of template ssODN or 1 µg targeting vector with assembled RNPs right before mixing with cells.
TITLE: Decreased risk of Alzheimer disease in bladder cancer patients with intravesical Bacillus Calmette-Guérin therapy: An exploratory meta-analysis
AUTHORS: Junjie Tian, Jianguo Gao, Xiaoyi Chen, Baiye Jin

[DESCRIPTION]
Alzheimer disease (AD) is the primary cause of chronic progressive dementia worldwide, accounting for about 60-80% of all cases of dementia. It is a progressive neurodegenerative disorder affecting 50 million people around the world, and the number will continue to increase in future with the aging of the population. The public health burden arising from AD is expected to increase dramatically in the coming decades according to demographic trends.
Recently, accumulating experiments showed that BCG treatment could prevent, delay, and even reverse cognitive decline in various animal models for AD. To date, several retrospective observational cohort studies have reported the influence of BCG use or not on AD incidence in bladder cancer patients, but with controversial results. So far, the efficacy of BCG on the risk of AD in bladder cancer patients has not been systematically evaluated yet. Hence, our current exploratory meta-analysis aimed at investigating the efficacy of BCG in reducing AD risk among bladder cancer patients, though the relatively limited number of high-quality studies are available. 

[STEPS]
1.
TITLE: Xenograft snRNA-seq Integration and Normalization
AUTHORS: Zac Chatterton, Chiara Pavan, Clare Parish

[DESCRIPTION]
This protocol describes the integration and normalization of single-nucleus RNA sequencing (snRNA-seq) data obtained from xenograft samples. The process involves loading data from multiple Cell Ranger outputs, creating Seurat objects, performing data integration, and normalizing the datasets. The protocol includes the visualization of results and statistical analyses to assess the quality and characteristics of the integrated datasets. The protocol includes the assignment of cell-type and differential gene expression analysis.

[BEFORE_START]
The user can access the code base at https://github.com/zchatt/ASAPSpatialTranscriptomics/tree/main/xenograft/snrna_seq

[STEPS]
SECTION: Setup and Data Loading:
1. Set the working directory and specify the path for Cell Ranger outputs.

Load the necessary R libraries and configure global options.
SECTION: Data Integration
2. Import multiple Cell Ranger outputs.

Create Seurat objects for each dataset.
SECTION: Quality Control (QC) and Data Normalization
3. Filter cells based on unique feature counts (e.g., remove cells with < 2000 unique counts or > 0.1% mitochondrial reads).

Integrate multiple datasets and normalize the data using Seurat’s standard workflow.
SECTION: Selection of Human Cells
4. Differentiate between human and rodent cells using per-cell read alignments to the respective genomes.
SECTION: Analysis and Visualization
5. Perform dimensionality reduction (e.g., PCA, UMAP) and clustering.

Visualize the integrated data using UMAP plots.
SECTION: Cell-Type Definition
6. Classify human cell clusters using tools like CHETAH and scType.
SECTION: Statistical Analysis
7. Conduct differential expression analysis between human cell types of interest.
TITLE:  Explainability of Foundation Models
AUTHORS: Paola Di Maio

[DESCRIPTION]
A metamodelling approach to support the development of a specifications for explainability in foundation models, adopting a combination of visualization, natural language representation.

[STEPS]
1. Establish Ground Truth
2. Define Alignment
4. Select Knowledge Representation 
5. Visualise KR
6. Black box testing
3. Define concept, key terms
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The research protocol on the topic:</p>
<p>Study of drug susceptibility of slowly growing nontuberculous mycobacteria (NTM) contains information on sample collection, processing diagnostic material, culture growth, primary differentiation of the isolated culture, and determination of mycobacterial species, drug susceptibility testing of slowly growing nontuberculous mycobacteria prevailing in Moscow region, MICs determination by Sensititre SlowMyco test.</p>

[STEPS]
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TITLE: SOP54v1_TGD_GenotypingSubmissionforCollaborators
AUTHORS: Varsha Rajesh

[DESCRIPTION]
This is the protocol to submit samples for genotyping for collaborator samples. 

[BEFORE_START]
QC Template:   


[STEPS]
1. Go to Stanford ilabs: https://stanford.ilabsolutions.com/landing/7
2. Log in with your credentials, then select Core Facilities in the left hand side menu. 
3. Select Stanford Genomics. Then select Request Services. 
 

4. Select "Whole Genome Genotyping Assay Service Request (Genotyping)" in their list of services. 
5. 
Fill out the first part of the form like so:


 

6. Fill out the second part of the form like so:

 

6.1. In Project Title, write the correct batch number of this genotyping batch. 
Upload the Genotyping QC file for the batch of samples you are submitting, in the "QC Results File" section. The template for this is here:   
Fill out the sample information form - you can download an excel template and then reupload to make it easier. 
7. In the payment section, fill out the PTA information. You will need to know the PTA breakdown and distribution for each sample type (each collaborator might require a different grant be used). Have that reflected here. 
 

 

8. Click submit request to core. 
9. Prepare your samples in a 96 well plate. Remember to indicate which sample went where in your QC spreadsheet, in plate layout. 
Dilute samples to 50 ng/uL in 15 uL of elution buffer. If samples are very low in concentration (below 10 ng/uL), provide the entire sample in a tube to the Stanford Genomics core, and indicate that it needs speed vacuuming in the QC spreadsheet.
TITLE: Luciferase Assay pH-dependent Fox-activity
AUTHORS: kyle.kisor

[DESCRIPTION]
Working protocol for how to perform luciferase assay. Worked for Fox reporter

[BEFORE_START]
Read entire protocol before starting

[STEPS]
SECTION: Plate Cells
1. 1. Plate 500K cells/well for each conditon
SECTION: Plate Cells
2. Incubate o/n before transfection
SECTION: Transfection
3. For each transfection condition create the following tube mixtures. (Volumes are for 1 transfection per condition and can be scaled up)
a. Tube 1: 125uL Optimem + 4uL lipofectamine 3000
b. Tube 2: 125uL Optimem + 2uL p3000 reagent + 1 ug total DNA
SECTION: Transfection
4. flick and incubate 5 minutes
SECTION: Transfection
5. Mix together tube 1 and tube 2 and incubate 15 min 

SECTION: Transfection
6. Add dropwise to cells and incubate o/n
SECTION: Transfection
7. Incubate 8 hours
SECTION: Transfection
8. Change media to fresh media or media + 10mM EIPA
SECTION: 48 hours incubation
9. Incubate cells for 48 hours after transfection. Change media at 24 hours post transfection with fresh media and fresh media + EIPA
SECTION: Luciferase Assay
10. Slowly thaw luciferase reagent in room temp water

SECTION: Luciferase Assay
11. Wash gently each well with PBS

SECTION: Luciferase Assay
12. Add 500uL of luciferase reagent directly to plate and nutate for 10 min RT

SECTION: Luciferase Assay
13. Scrape each well with cell scraper and put into fresh microfuge tube

SECTION: Luciferase Assay
14. Spin down 13,000 rpm 5 min at RT 

SECTION: Luciferase Assay
15. Add 100uL of supernatant to 4wells of white 96well plate. Wait 10 minutes

SECTION: Luciferase Assay
16. Read luciferase on plate reader

SECTION: Luciferase Assay
17. For Renilla reading, calculate reagent needed for 100uL each well using a 1:100 dilution of reagent: buffer 
SECTION: Luciferase Assay
18. Add 100uL of renilla reagent to each well and wait 10 min before reading
TITLE: CAMbank: cfDNA BCT Field Processing v1
AUTHORS: Eliah G Overbey, Krista A Ryon, JangKeun Kim, Christopher E Mason

[DESCRIPTION]
Field processing of cfDNA BCTs for the Cornell Aerospace Medicine Biobank (CAMbank).

Instructions for preserving: plasma and RBC Pellets.

[STEPS]
SECTION: Perform Venipuncture
1. After venipuncture, invert the tubes gently 8 to 10 times to fully mix tube anticoagulant with blood sample.

Store the tube upright at room temperature until centrifugation.

Note: The tube stabilizes cell-free DNA for up to 14 days at 6 °C to 37 °C and CTCs for up to 7 days at 15 °C to 30 °C.
SECTION: Centrifuge Settings: Plasma Separation
2. Note: A swing bucket centrifuge is required. 

Set centrifuge:
acceleration: 9
deceleration: 9
temperature: RT
duration: 20 minutes
speed: 300xg
SECTION: Centrifuge Settings: Plasma Separation
3. Place the cfDNA BCTs in the centrifuge. 
Place a protective cover over the swing buckets in case of tube leakage. 
Start the centrifuge. 
Stand by the centrifuge until the centrifuge reaches max speed. Listen for signs of imbalance or compromised tube integrity. 
SECTION: Separate Plasma
4. Carefully remove cfDNA BCTs from the centrifuge and inspect for separation of red blood vs plasma layers. 

Transfer cfDNA BCTs to a sanitized laminar flow hood. 
SECTION: Separate Plasma
5. Remove the upper plasma layer and transfer to a new 15mL conical tube. 
Cap the conical tubes and return to the centrifuge. 
SECTION: Aliquot Plasma
9. Carefully remove conical tubes from centrifuge. 
Transfer conical tubes to a sanitized laminar flow hood. 
SECTION: Aliquot Plasma
10. Using a P1000, carefully aliquot plasma into 2D barcoded tubes at 500µL each. 

Return 2D tubes with plasma to the rack and place in the -80C freezer.
SECTION: Centrifuge Settings: Plasma Purification
6. Note: A swing bucket centrifuge is required. 

Set centrifuge:
acceleration: 9
deceleration: 9
temperature: RT
duration: 10 minutes
speed: 5000xg
SECTION: Centrifuge Settings: Plasma Purification
7. Place the conical tubes in the centrifuge. 
Place a protective cover over the swing buckets in case of tube leakage. 
Start the centrifuge. 
Stand by the centrifuge until the centrifuge reaches max speed. Listen for signs of imbalance or compromised tube integrity. 
SECTION: Freeze Red Blood Cell Pellets
8. Recap and freeze the cfDNA tubes to preserve DNA in the red blood cell pellet
TITLE: Processing ddRAD data from raw fastqs to vcf
AUTHORS: Thom Nelson, Findley Finseth, Lila Fishman, Colette Sevier Berg

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a pipeline that the Fishman lab uses for processing paired-end ddRAD data, from fastq to vcf. These scripts were written and pipeline developed by Findley Finseth and Thom Nelson. </div></div>

[STEPS]
?. [Remove PCR duplicates using molecular barcode]
This step removes PCR duplicate using the i5 molecular barcode.The resulting files will have the same prefix, but will have the suffix .rmdup.1.fastq (forward reads) and .rmdup.2.fastq (reverse reads)
?. [Demultiplex plates using i7 barcode]
This step requires 4 fastq files: forward reads, reverse reads, the i7 file, and the i5 file, and then a barcode file. The output files will be demultiplexed fastq files with the prefix from the first column of the barcode file.  Each half-plate will have a forward and reverse read.
Sample barcode file:plate_name  \t i7 barcode
?. [Flip the reads]
Use this command to flip the reads for each half-plate. The output file will be called filtered_forward.fastq and filtered_reverse.fastq, so now is a good time to make separate folders for each half-plate and put the resultng files in those folders.
The barcode file is a list of 48 RAD barcodes used.
?. [Demultiplex by individual; join forward and reverse reads]
This uses the process_shortreads bash command to demultiplex wach half-plate into one file for each individual. The input files required are filtered_forward.fastq, filtered_reverse.fastq, and a barcode file. The outputs will be zipped fastqs for each individual, with the suffix .fq.gz
Barcode file format:barcode \t sample_nameDouble and triple check that the barcodes correspond to the correct sample name!
?. [Make bams]
This script uses the tools GATK and Trimmomatic, so those tools need to be installed beforehand. The input files for this script are the sample fastq and the genome FASTA file. The genome file also needs to have the following index files: .dict, .fa.amb, .fa.bwt, .fa.fai, .fa.pac, .fa.sa with the same prefix as the genome file & stored in the same directory as the genome. This will output a .bam and a .bai for each individual. Now is a good time to to QC by visually examining the bams in IGV
?. [Make genomic vcfs (gvcfs)]
This step takes bams and makes a gvcf for each individual using the attached script, then combines them all into a vcf. Put all the bams into one directory first, and then edit the script so that there is a path to the directory with all the bams.
TITLE: CBM for propagation of in vitro plantlets
AUTHORS: Lynn    Doran, Amanda  P. De Souza

[DESCRIPTION]
Media preparation for in vitro propagation of plantlets.  Validated for cassava in tissue culture.

[GUIDELINES]
Thoroughly sterilize all materials and work surfaces before pouring media.
Use sterile technique after autoclaving, when pouring media into containers.  
Wear a lab coat and clean gloves that have been thoroughly soaked in 70% ethanol during media pouring.
Do not turn off laminar flow hood blower fans during media setting time.
Do not leave UV light on once media is in the flow hood.  The UV light will damage the vitamin content of the media.

[STEPS]
TITLE: ClearLab Flu+COVID19 Antigen Assay IFU
AUTHORS: petercb3 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"> Indication:  Qualitative detection of the nucleocapsid protein antigen from SARS-CoV-2 in nasopharyngeal aspirate specimens directly or after the aspirate have been added to viral transport media from individuals who are suspected of COVID-19 by their healthcare provider. Emergency use of this test is limited to authorized laboratories and other authorized testing locations.</div></div>

[STEPS]
?. Collect specimen with Clearinse Nasal Wash Aspiration System
?. Pipette 100 uL out from specimen chamber (up to first mark of graduated transfer pipette)
?. Dispense entire sample from pipette into the assay cassette's extraction well.
?. Then, pipette 50uL reagent into the assay cassette's extraction well and let sit for one minute.
?. Raise assay cassette upright and let stand for 2 seconds
?. Lay device back down and read results in 10 minutes.