eamag/protocols-with-steps huggingface dataset:
TITLE: Protocol for RNA isolation and RT‐PCR confirmation for Neomycin gene expression in transformed Bodo saltans
AUTHORS: Fatma Gomaa, Zhu-Hong Li, Roberto Docampo, Virginia Edgcomb

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Developing transfection protocol for </span><span style = "font-style:italic;">Bodo saltans</span><span>, using SaCas9/sgRNA ribonucleoprotein (RNP) complex in conjunction with DNA repair template to disrupt the Paraflagellar rod 2 gene (</span><span style = "font-style:italic;">BsPFR2</span><span>) and increase the efficiency of targeted homologous recombination when a repair template DNA is provided. The exogenous repair template is double stranded DNA and it consists of </span><span style = "font-style:italic;">eGFP</span><span> fused with the drug selection gene </span><span style = "font-style:italic;">nptII/neo </span><span>and flanked by 500 bp of the untranslated regions (UTRs) upstream and downstream of the targeted </span><span style = "font-style:italic;">BsPFR2</span><span> as homologous repair arms.</span></div><br/></div>

[STEPS]
?. -Total RNA was isolated from 4 months old transformed clones and wild type Bodo saltans cultures using the Quick-RNA Miniprep Kit (Zymo Research) according to the manufacturer’s instructions.
?. -RNA underwent two rounds of DNAse treatments. First, we performed an on-column DNAse digestion, following the protocol of the Quick-RNA Miniprep Kit (Zymo Research). Second, DNAse digestion was performed on the purified RNA using gDNA wipeout buffer and the QuantiTect Reverse Transcription Kit (Qiagen, Germany), in the following 14 µl reaction: 2 µl of gDNA wipeout buffer 7x, 6 µl of RNA, and 6 µl of RNase-free water.
?. -cDNA was synthesized using the QuantiTect Reverse Transcription Kit (Qiagen, Germany) according to the manufacturer’s instructions, the RT Primer Mix and the 5X Quantiscript RT buffer were mixed in 1:4 ratio in a 20 µl reaction that included 14 µl of RNA template (post-DNA elimination).
?. -cDNA was then amplified in a 25 µl PCR reaction, with primers targeting  the Neomycin gene; the Neo F primer (5’-CGCTTGGGTGGAGAGGCTATTCGGCTATGAC 3’) and Neo R primer (5’-TCCACCATGATATTCGGCAAGCAGGCATC -3’).
?. -Control PCR reactions was performed using RNA template without the RT to verify the absence of the DNA from the transformed B. saltans cell.
?. -PCR products were visualized by gel electrophoresis, with purified plasmid as a positive control for the PCR. Amplified PCR products at the expected size of 550-pb were documented (Figure 1).
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Histones are a family of basic proteins that associate with DNA in the nucleus and help condense it into chromatin, they are alkaline (basic pH) proteins, and their positive charges allow them to associate with DNA. They are found inside the nucleus of eukaryotic cells. DNA and histones are packed together to be nucleosome, nucleosome form a pack which are called chromatin, two chromatin form a chromosome.</p>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Frost formation on solid surfaces is ubiquitous in nature when the water vapor droplets contact surfaces with low temperature. The protocol is for carrying out the frost on the butterfly wing surface and analyzing the hydrophobic durability mechanism of butterfly wing surface. The protocol has been tested on ten different kinds of the butterfly species, <em>Papilio maackii, Gonepteryx mahaguru, Papilio xuthus, Vanessa cardui, Pieris napi, Argynnis paphia, PierisrapaeLinne, Parnassius glacialis, Mimathyma nycteis, Danaus genutia.</em></p>

[BEFORE_START]
<p>The microscopic observation and recording apparatus should be carried out many times of freezing test to testify the stability of cooling surface temperature.</p>
<p>Use a solution with a stock time of less than 3 months.</p>

[GUIDELINES]
<p>The frosting process on wing surface of different butterfly species are observed by using self-designed microscopic observation and recording apparatus.</p>
<p>Atomic force microscopy (AFM) is used to observe the microstructure of butterfly wing surface, and measure the distance between the adjacent longitudinal ribs.</p>
<p>The contact angle of butterfly wing surface is measured by adopting the contact angle measurement system (OCA 30-2).</p>

[STEPS]
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TITLE: Isolation of endothelial cells from umbillical vessels
AUTHORS: Thomas Mudersbach, Daniel Siuda, Karin Kohlstedt, Ingrid Fleming

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

[STEPS]
?. [Reagents]
Hank's Balanced Salt Solution (HBSS) (H6648-500ml, Sigma-Aldrich)Enzymatic digestion with Dispase II (29585300, Roche) (2.4 U/ml) in Ham's F12Ham's F12 with 10% fetal calf serum (FCS), penicillin (50 U/ml) and streptomycin (50 µg/ml) for centrifugationEndothelial cell culture medium: MCDB131 (10372-019, Gibco) containing 10% FCS, L-glutamine (10mM), penicillin (50 U/ml), streptomycin (50 µg/ml), epidermal growth factor (0.1 ng/ml), basic fibroblast growth factor (1 ng/ml) and endothelial growth supplement with Heparin (0.4%)
?. [Endothelial cell isolation]
umbilical cord is cut at one end (about 1 cm) to identify the arteries and the veinthe vein or artery is then cannulated with a button cannula connected to a three-way stop cock. The cannula is fixed with a cable connector to the cordThe cord is the rinsed with HBSS until the HBSS remains clear and no blood is washed out any longerRepeat steps 1-3 on the other endNow both ends are cannulated and the vessel is filled with Dispase II solutionThe cord is incubated for  at  Discard the Dispase II solution and replace with Ham's F12 containing 10 % FCSCarefully massage the cord to loosen the endothelial cellsThe cord is incubated for at Discard the Dispase II solution and replace with Ham's F12 containing 10 % FCSCarefully massage the cord to loosen the endothelial cellsCollect the medium in a suitable centrifugation tube and centrifuge for at  100 x gDiscard the supernatant and resuspend pellet in endothelial cell culture medium and distribute on a fibronectin-coated cell culture dish. Coating with fibronectin should be performed  to before use. Place the cell culture dish containing your cells in a cell incubator atin 5% CO2.After cells are washed with cell culture medium. to after isolation the cells should have formed a confluent cell layer
[min]
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[min]
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[hr]
TITLE: Calcium phosphate transfection mammalian cells
AUTHORS: Karla Feijs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is our calcium phosphate transfection protocol. It always works with HEK293T cells, for other lines the transfection efficiency depends on their source: some HeLa and U2OS clones can be transfected using CaPO4, however most cannot. We mostly use this method to transfect HEK293T cells for lentivirus production.</div></div>

[STEPS]
?. [Day 1]
Seed cells: density depends on cell line and dish used. For example in a 10cm dish seed 0.8x106 HeLa or 1.5 x106 HEK293T cells; in a 6-well plate seed 0.3 x106 HEK293T cells or 0.15x106 HeLa cells.
?. [Day 2]
Observe the cells under the microscope.Prepare the transfection mix. The amounts given here are for a 10cm dish and can be adjusted to other dishes and plates.Prepare in an eppendorf tube:Add Add
[plasmid DNA]
[HEBS buffer]
[ CaCl2]
We rarely use a full 20µg of DNA encoding for something: some times as little as 2 µg of the total is encoding for a protein of interest, whereas the rest is an empty vector. This amount of DNA is required to efficiently form precipitates.
?. [Day 2]
Add the mixture drop-wise onto the cells, gently agitate the dish to evenly distribute the mixture and place the cells back in the incubator for 5-6 hours ().
?. [Day 2]
After 5-6 hours, remove the media, add warm HEPES buffer and incubate 5-10 minutes ().
?. [Day 3]
Day 3-4Process your cells 24 to 48 hours after transfection, incubation time depends on experimental needs and set-up. If it does not interfere with the experiment, 5% of the transfected plasmid DNA can be a GFP-encoding plasmid to monitor transfection efficiency.
?. [Day 2]
Flick the tube gently to thoroughly mix and incubate at  for approximately .
0 Room temperature
?. [Day 2]
Remove the HEPES and add fresh full DMEM. The precipitates are visible under a microscope and should disappear during the incubation with HEPES. Replace the cells in the incubator.
TITLE: Immunohistochemistry data processing
AUTHORS: Tae-Un Han

[DESCRIPTION]
This protocol has been used for high throughput quantification of immunohistochemistry data of mouse brain sections

[STEPS]
1. Widefield fluorescent images are collected for GFP and DAPI fluorescent channels using a Zeiss AxioScan.Z1 slide scanning microscope system with a Plan-Apochromat 20x/0.8 objective lens.
2. All images are acquired using a Hamamatsu Orca flash 4.0 camera with an average tile count of 165 tiles per brain section.
3. The Zeiss ZEN blue 2.3 software package was used for collection and stitching of the 2-color (DAPI & GFP) tiled images.
4. Widefield fluorescent images are then post-processed using MediaCybernetics’ Image-Pro 11 software package (Rockville, MD, USA).
5. Every stitched image is processed using a protocol modified for each antibody. Initially, the image is masked to solely include the tissue in areas of interest. 
7. Smart Segmentation is used to separate GFP-expressing puncta or fibrils from DAPI-stained nuclei in tissue evaluated with each antibody.
6. Threshold segmentation is used for each antibody staining to separate actual signal from background auto-fluorescence. 
8. The Count/Size function is designed to extract the percent of the tissue sample that stains positive for GFP.
TITLE: Intracardial perfusion for electrophysiology in rat
AUTHORS: Alicia Avelar

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is to use for clearing blood from the brain and helping to maintain viable cells for electrophysiology experiments.  No PFA or any tools that have had contact with PFA should be used. This intracranial perfusion can help maintain viability of neurons in older animals and those who have undergone treatments that damage brain cells (for example: chronic methamphetamine and Parkinson's disease like treatments). </div></div>

[STEPS]
?. [Anesthesia]
anesthetize rat with isoflurane anesthesia machine.
?. [Anesthesia]
when rat is lying down immobile, doesn’t right it’s position when moved, and is breathing slowly put isoflurane nose cone on rat and test for pain with forceps toe pinch of both feet
?. [Surgery and intracranial perfusion]
open rat thoracic cavity under sternum
?. [Surgery and intracranial perfusion]
cut diaphragm
?. [Surgery and intracranial perfusion]
cut sides of rib cage
?. [Surgery and intracranial perfusion]
hold sternum back with forceps
?. [Surgery and intracranial perfusion]
small cut to open right atria
?. [Surgery and intracranial perfusion]
inject into heart ventricle ~ 120 mL ice cold artificial cerebral spinal fluid, then ~ 120 mL ice cold high sucrose solution
?. [Brain extraction]
decapitate rat
?. [Brain extraction]
carefully remove skull
?. [Brain extraction]
gently free brain from skull, cut cranial nerves when necessary
?. [Brain extraction]
let brain gently release from skull into ice cold high sucrose solution
?. [Cleanup]
clean surgical tools carefully and let air dry
?. [Cleanup]
sharps disposed of in sharps biohazard red box
?. [Cleanup]
let blood dissolve in bleach in flask in hood to decontaminate
?. [Cleanup]
dispose of other biohazard material in a double bagged biohazard red bag .add sticker label. dispose of in biohazard waste bins.
?. [Cleanup]
clean surfaces in hood with ethanol and paper towels
?. [Cleanup]
bring rat body in body bag with labeled tag to BSB 099 cold room
?. [Cleanup]
bring cage and accessories with it to BSB dirty cage area for washing
0 µl
TITLE: Making slip
AUTHORS: Amber  York

[DESCRIPTION]
Rough procedure for making slip including colored slip.

[STEPS]
1. Bone dry clay (small chunks/trimmings are better).  Drying clay first allows it to break down and mix better than using wet recycled clay.
2. Cover with water. Soak overnight at least.
3. Blend.  I used an old blender.  Adding water as needed to keep it moving.
4. Add colorants. 

5. Blend
6. If using as slip, keep it in a sealed container.
7. If using as moist clay, pour onto plaster to dehydrate.
4.1. To keep track of the percent colorant you first have to get the amount of clay in your slip (vs water content).
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The immunostaining detection of isolated lymphoid follicles (ILFs) was performed according to the method of McDonald and Newberry [BioTechniques. 2007; 43(1): 50-56], with some modifications. Instead of mounting the tissue samples on a mounting plate, each segment was treated in a 15 ml tube in each step. DAB stained intestines were finally mounted on a glass slide using an aqueous mounting medium.</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p style="text-align: justify;">Ancient DNA research has been revolutionized following the development of ‘Next Generation’ Sequencing platforms. Although a number of such platforms have been applied to ancient DNA samples, the Illumina series are the dominant choice today, mainly because of high production capacities and short read production. Recently a potentially attractive alternative platform for palaeogenomic data generation has been developed, the BGISEQ-500, whose sequence output are comparable with the Illumina series. In this study, we modified the standard BGISEQ-500 library preparation specifically for use on degraded DNA, then directly compared the sequencing performance and data quality of the BGISEQ-500 to the Illumina HiSeq2500 platform, on DNA extracted from eight historic and ancient dog and wolf samples.&lt;br&gt; The data generated was largely comparable between sequencing platforms, with no statistically significant difference observed for parameters including level (p=0.371) and average sequence length (p=0718) of endogenous nuclear DNA, sequence GC content (p=0.311), double-stranded DNA damage rate (p=0.309), and sequence clonality (p=0.093). Small significant differences were found in single strand DNA damage rate (δS, slightly lower for the BGISEQ-500, p=0.011) and the background rate of difference from the reference genome (θ, slightly higher for BGISEQ-500, p=0.012). This may result from the differences in amplification cycles used to PCR amplify the libraries. A significant difference was also observed in the mitochondrial DNA percentages recovered (p=0.018), although we believe this is likely a stochastic effect relating to the extremely low levels of mitochondria that were sequenced from three of the samples with overall very low levels of endogenous DNA. &lt;br&gt; Although we acknowledge our analyses were limited to animal material, our observations suggest that the BGISEQ-500 holds the potential to represent valid and potentially valuable alternative platform for palaeogenomic data generation, that is worthy of future exploration by those interested in the sequencing and analysis of degraded DNA.</p>

[STEPS]
TITLE: Isolation of rodent eyes for analysis of diabetic retinopathy
AUTHORS: Timothy Kern

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">This protocol describes the procedure used by the DiaComp to isolate rodent eyes to phenotype for Diabetic Retinopathy. </div><div class = "text-block"><span style = "font-weight:bold;">Diabetic Complication:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bhxk4kcn.jpg" /></div></div>

[STEPS]
?. What we want: Both eyes in formalin from at least 5 animals per experimental group. The longer the duration of diabetes, the better (C57BL/6 mice require at least 6 months diabetes before we can detect retinal microvascular lesions). Include also age-matched nondiabetic animals and diabetic controls for comparison.
?. Eye collection All tissue needs to be collected fresh (within 10-15 min of death) to get best results. NOTE: Tissue post-mortem more than 1 hour does not work! • Anesthetize animal (we have not yet found that any particular anesthetic is better or worse than others). • Place thumb and pointer finger on either side of eye. By spreading thumb and pointer finger, stretch the skin around the eye. • Using fine, sharp scissors, make 3-5 deep (5-10mm) cuts while forcing scissors into the eye socket. The eye now should be attached only by the optic nerve leading into the brain. Push the scissor back into the eye socket again to  cut the nerve, so that there is some optic nerve still attached to the eye. Do not push on the skull to protrude the eye  and then cut horizontal across the skull; this can damage the eye. Be careful not to push or compress the eye.   See Figure Below • Drop both eyes into 10% buffered formalin (pH 7.4) in a microfuge tube. Variances in pH seem to influence how well the tissue can be stained later, so check the pH (especially if the fixative is prepared in your lab). Label each tube with animal number. Also send a summary sheet listing all animals and experimental description with date of death; please put your name and email address on the sheet as well. Group identifications need not be on this sheet until analysis is completed. Parafilm each tube to minimize the likelihood of leaks, and put tubes in a zip-lock bag. • We will need to receive both eyes within about 4 days of death and fixation if apoptosis is to be assessed (if apoptosis is not to be assessed, the duration in formalin is not important). Please give me a few days warning that tissue about when tissue will be shipped so that we can watch for it. Email tracking number once it is sent.
TITLE: Slot-blot analysis of recombinant α-synuclein fibrils
AUTHORS: Arpine Sokratian

[DESCRIPTION]
This protocol aims to provide an accurate analysis of aggregated species of α-synuclein. The method involves the immunoblotting of protein samples via low vacuum-induced filtration, followed by the blocking of the membrane and the attachment of antibodies specific to the protein. The procedure is designed to visualize the protein of interest in a slot-blot manner, which is suitable for intensity calculations. A critical aspect of this protocol is the correct setup of the slot-blot instrument to ensure equivalent vacuum pressure in each well of a 48-well setup. This step is crucial to guarantee the reproducibility and reliability of the results. The protocol is used to visualize and calculate the α-synuclein fibrils from cell tissues or those that are recombinantly expressed. Notably, the proteins are not denatured during the process, allowing for their capture in a physiological state

[STEPS]
SECTION: Sample preparation
4. Dilute samples to the final concentration of fibrils - 1 ug/mL. 
SECTION: Bio-dot slot format microfiltration apparatut assembly
5. Clean and dry the Bio-Dot apparatus and gasket prior to assembly

 



SECTION: Immunnoblotting
8. Place the membrane into the rack with 20 mL of TBS with 5% non-fat milk    
Block for 1 hour with continuous shaking at room temperature200 ul of samples in TBST. 
SECTION: Sample preparation
1. 5 mg/mL of sonicated fibrils (QC by DLS and nanodrop) were prepared in PBS
Chemical denaturants 



 

SECTION: Sample preparation
2. Prepare solutions of sonicated fibrils mixed with desired concentration of the denaturant, 
for example: 0.1 % SDS in 0,5 mg/mL sonicated fibrils solution, 0.01% SDS, 0.001% and etc.
SECTION: Sample preparation
3. Incubate solutions for 30 minutes (along with control samples prepared in PBS) at room temperature
SECTION: Bio-dot slot format microfiltration apparatut assembly
6. Insert previously soaked in TBS 0.22um nitrocellulose membrane using a bio-dot slot format microfiltration apparatus
 

SECTION: Bio-dot slot format microfiltration apparatut assembly
7. Add 200 ul of TBS to 48 chambers in the apparatus. Apply the low-speed vacuum to filter the liquid through the membrane. 
Add 200 ul of the fibril samples, the blank well should be filled with TBS, then apply the vacuum to fully drain the membrane.
SECTION: Immunnoblotting
9. Add the desired antibodies. In case to detect fibrilllar a-syn species add 1:5000 in TBST  

Incubate for 2 hours
SECTION: Immunnoblotting
10. Wash with TBST three times
SECTION: Immunnoblotting
11. Image Blot on BioRad Chemidoc
TITLE: CuPCR SARS-Cov-2    
AUTHORS: B Ronacher, Christoph Reschreiter

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">RNA based diagnostic test of different human samples (nasopharyngeal swabs, saliva) to detect SARS-Cov-2. The test requires RNA/DNA extraction and a follow-up RT-PCR (reverse transcriptase polymerase chain reaction). The test covers the N-gene and RdRp-gene and covers a human control as well. The human control safeguards the validity of sample taking and test procedure. </div></div>

[STEPS]
?. [RT-PCR test]
RNA / DNA extraction
100µl eluation buffer (e.g. water) containing RNA / DNA from the sample
?. [RT-PCR test]
RT-PCR
Prepare mastermix: 5µl of primer mix + 5µl of enzyme mix (per sample)Put mastermix and sample into PCR tube:10µl of mastermix + 10µl of sample (eluate)Select (or program) RT-PCR protocol:    □ 2 minutes 50 °C    □ 2 minutes 95 °C     □ 45 cycles:             5 seconds 95 °C            1 minute 60 °C            Scan    □ 25°C for hold Load samples and start PCRAnalyse amplification curves:    Human control:                ct  valid    N-gene SARS-Cov-2:        ct > 0 --> positive    RdRp-gene SARS-Cov-2:  ct > 0 --> positive
Amplification curves for: human control, N-gene of SARS-Cov-2, RdRp-gene of SARS-Cov-2
?. [RT-PCR test]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
MALDI-TOF MS specimen preparation for rapid identification of Australian mosquitoes

[GUIDELINES]
<p>There are 3 main steps involved with preparing and launching a mosquito sample for MALDI-TOF analysis:</p>
<ul>
<li>Preparing the matrix</li>
</ul>
<p>The matrix solution ionises the proteins in the sample and is applied to the plate after the sample. The matrix must be very acidic.</p>
<ul>
<li>Dissecting and crushing the sample</li>
</ul>
<ul>
<li>Preparing and launching the plate</li>
</ul>

[STEPS]
SECTION: Preparing the matrix
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SECTION: Preparing the matrix
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SECTION: Preparing the matrix
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SECTION: Preparing the matrix
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SECTION: Preparing the sample
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SECTION: Preparing the sample
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SECTION: Preparing the sample
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SECTION: Preparing the sample
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SECTION: Preparing the sample
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SECTION: Preparing the sample
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SECTION: Preparing the sample
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SECTION: Preparing the plate
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SECTION: Preparing the plate
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SECTION: Preparing the plate
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SECTION: Preparing the plate
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