eamag/protocols-with-steps huggingface dataset:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes the preparation of Illumina libraries for high through-put sequencing of near full-length bacterial 16S rRNA sequences. The method is based on the universal 27F and 1492R primers designed by Lane <em>et al</em>.*</p>
<p> </p>
<p>Sequences for primers and adaptor used can be found under guidelines.</p>
<p> </p>
<p style="margin-left: 24pt; text-indent: -24.0pt;">*Lane, D. J. (1991). 16S/23S rRNA sequencing. In E. Stackebrandt &amp; M. Goodfellow (Eds.), <em>Nucleic Acid Techniques in Bacterial Systematics</em> (pp. 115–175). Chichester, United Kingdom: John Wiley and Sons.</p>
<p> </p>

[GUIDELINES]
<p><strong>Primers and adaptors used for library preparation:</strong></p>
<table width="956">
<tbody>
<tr>
<td width="154">Oligo name</td>
<td width="738">sequence</td>
<td width="64">Purification</td>
</tr>
<tr>
<td>B16S_rDNA_PCR1_fw1</td>
<td>CTCCACCCAGACTCATCCATNNNNNNNNNNNNNNNTGCCTCTTAGAGTTTGATCMTGGCTCAG</td>
<td>PAGE</td>
</tr>
<tr>
<td>B16S_rDNA_PCR1_fw2</td>
<td>CTCCACCCAGACTCATCCATNNNNNNNNNNNNNNNTCCTCTACAGAGTTTGATCMTGGCTCAG</td>
<td>PAGE</td>
</tr>
<tr>
<td>B16S_rDNA_PCR1_fw3</td>
<td>CTCCACCCAGACTCATCCATNNNNNNNNNNNNNNNTCATGAGCAGAGTTTGATCMTGGCTCAG</td>
<td>PAGE</td>
</tr>
<tr>
<td>B16S_rDNA_PCR1_fw4</td>
<td>CTCCACCCAGACTCATCCATNNNNNNNNNNNNNNNCCTGAGATAGAGTTTGATCMTGGCTCAG</td>
<td>PAGE</td>
</tr>
<tr>
<td>B16S_rDNA_PCR1_fw5</td>
<td>CTCCACCCAGACTCATCCATNNNNNNNNNNNNNNNTAGCGAGTAGAGTTTGATCMTGGCTCAG</td>
<td>PAGE</td>
</tr>
<tr>
<td>B16S_rDNA_PCR1_fw6</td>
<td>CTCCACCCAGACTCATCCATNNNNNNNNNNNNNNNGTAGCTCCAGAGTTTGATCMTGGCTCAG</td>
<td>PAGE</td>
</tr>
<tr>
<td>B16S_rDNA_PCR1_rv</td>
<td>AGGGGGGCAAAGATGAAGATNNNNNNNNNNNNNNNCGTACTAGTACGGYTACCTTGTTACGACTT</td>
<td>PAGE</td>
</tr>
<tr>
<td>B16S_rDNA_PCR2_fw</td>
<td>CTCCACCCAGACTCATCCAT</td>
<td>PAGE</td>
</tr>
<tr>
<td>B16S_rDNA_PCR2_rv</td>
<td>AGGGGGGCAAAGATGAAGAT</td>
<td>PAGE</td>
</tr>
<tr>
<td>B16S_rDNA_readtag_rv </td>
<td>CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAGGGGGGCAAAGATGAAGAT</td>
<td>PAGE</td>
</tr>
<tr>
<td>B16S_rDNA_readtag_fw</td>
<td>CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTCCACCCAGACTCATCCAT</td>
<td>PAGE</td>
</tr>
<tr>
<td>B16S_rDNA_linktag_fw</td>
<td>CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCTCTGAGCCAKGATCAAACTCT</td>
<td>PAGE</td>
</tr>
<tr>
<td>B16S_rDNA_linktag_rv</td>
<td>AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTAAGTCGTAACAAGGTARCCGTA</td>
<td>PAGE</td>
</tr>
<tr>
<td>B16S_rDNA_read2_fw</td>
<td>GCTCTTCCGATCTCTCCACCCAGACTCATCCAT </td>
<td>HPLC</td>
</tr>
<tr>
<td>B16S_rDNA_read2_rv</td>
<td>GCTCTTCCGATCTAGGGGGGCAAAGATGAAGAT </td>
<td>HPLC</td>
</tr>
</tbody>
</table>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Copy of          ELISA for quantification of human C5 in serum or plasma.
AUTHORS: Angel  Justiz-Vaillant, Belkis Ferrer-Cosme

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

[STEPS]
?. An anti-human C5 coating antibody is adsorbed onto the microwells by incubation overnight at 4°C with carbonate-bicarbonate buffer.
?. Add 50 µl of human serum or plasma. Human C5 present in the serum or plasma binds to antibodies adsorbed into the microwells.
?. The microplate is blocked with 3% non-fat milk-PBS buffer and later wash to remove unbound proteins.
?. Fifty (50) µl of biotin-conjugated anti-C5 antibody is added. The optimal dilution must be investigated.
?. The microplate is rewashed with PBS-Tween 20 buffer, pH 7.4.
?. One hundred µl of streptavidin-HRP conjugate is added and it binds to the biotin-conjugated anti-C5 antibody. The optimal dilution of this conjugate must be investigated.
?. The plate is washed following incubation to remove the unbound Streptavidin-HRP.
?. Add 100 µl of 3,3',5,5'- tetramethylbenzidine (TMB; Sigma-Aldrich) into each well.
?. Incubate the microwells in the dark for 15 min.
?. A colored product is formed in proportion to the quantity of C5 present in the sample or standard.
?. The reaction is terminated by addition of 100 µl 3M H2SO4  and the absorbance is measured at 450 nm.
?. A standard curve is made from 7 human C5 standard dilutions and the human C5 sample concentration is determined.
TITLE: Operating the gassing manifold
AUTHORS: Roey Angel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Standard operating procedure for using the gassing manifold</span></div><div class = "text-block"><span>The gassing manifold is used for replacing the headspace of vials with N</span><span style = "vertical-align:sub;">2</span><span> (or N</span><span style = "vertical-align:sub;">2</span><span>:CO</span><span style = "vertical-align:sub;">2</span><span>) or for degassing solutions. It is useful for preparing anoxic incubations, media and buffers before bringing them in closed vials into the anoxic chamber. The manifold is equipped with an electronic controller that automatically switches between the gas inlet and the vacuum pump and performs multiple cycles of headspace flushing.</span></div></div>

[STEPS]
?. Attach new needles to the syringe filters. If working with sensitive samples, the syringe filters should also be changed to avoid contamination.
The selected needles must be thick enough to penetrate the stoppers, but thin enough as to not break the stoppers.
?. Attach the vials by piercing the stoppers with the attached needles.
The needle should never touch the liquid inside the bottle!
?. Turn on the main pressure knob almost all the way through.
Never force the knob open or close beyond its stopping point!
?. Turn on the pressure adjusting knob. Usually this is set to be between 0.5 and 1 bar.
Never force the knob open or close beyond its stopping point!Whatever the pressure gauge shows will also be the level of overpressure in the vial!
?. Turn on the pump.
?. Open the valves of the tubes to which vials were connected.
Keep all other valvles closed.
?. Flip the switch to turn on the controller.
?. Wait and allow the vials to be flushed properly.
The number of flushing cycles needed to turn the vials anoxic will depend on their content. Empty vials only need a about 3-5 cycles to become anoxic but vials with liquids will require longer to degas completely. This should be tested when using a new set-up of vials.
?. Turn off the controller switch and allow the vials to reach overpressure.
What the pressure gauge above the controller shows is the overpressure in the vials
?. Turn off the pump.
?. Close the vavles and remove the vials.
?. Turn off the main pressure knob.
Do not use excess force!
?. Turn off the main pressure adjusting knob.
Do not use excess force!
?. Release the remaining pressure in the system by quickly opening and closing one of the tube valves.
TITLE: Sea Water - Blue Treasure
AUTHORS: Willian Barela Costa

[DESCRIPTION]
Sea water production for microalgae cultivation using the Blue Treasure supplier. 

25 - 35 Salinity

27 mg/mL 



[STEPS]
SECTION: Salting process - 1 liter
1. 0.027 g/mL ->   27 g Sea Sal to 1 L     

SECTION: Salting process - 1 liter
2. Shake until diluted
SECTION: Salting process - 1 liter
3. Make sure the salinity is within the desired range using the salinity refractometer. In this protocol, the focus is 27 salinity.
TITLE: Plague in Zimbabwe from 1974-2018 A Review Article
AUTHORS: AMON MUNYENYIWA, Moses Zimba, Tamuka Nhiwatiwa, Maxwell Barson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The protocol describes how literature was searched to write a review Article titled :Plague in Zimbabwe from 1974-2018.Literature review was conducted using the online databases PubMed, and HINARI. Hard copy text books with information relevant to plague were obtained from the University of Zimbabwe library.The search was refined to manuscripts that reported the occurrence of animal or human plague in Southern, Central, Eastern, Northern Africa and the whole world. All the information was supplemented with information from Web-accessible documents and other grey literature from libraries and archives.</div></div>

[STEPS]
?. Materials and Methods
?. The literature review was conducted using the online databases PubMed, and HINARI; Hard copy textbooks with information relevant to plague were obtained from the University of Zimbabwe library.
?. A thorough search of work was then undertaken in Zimbabwe from the first case of plague in 1974 in Hwange National Park to recent findings.
?. The work includes dissertations and unpublished reports from the Ministry of Health and Child Care, with particular emphasis on the epidemiology of plague in Zimbabwe.
?. A systematic search strategy was employed using the following search terms: ‘Africa’ or the name of any African country (e.g. ‘Rhodesia or Zimbabwe and ‘plague’ (e.g. ‘Yersinia pestis’).
?. The search was refined to manuscripts that reported the occurrence of animal or human plague in Southern, Central, Eastern, Northern Africa, and the whole world.
?. All the information was supplemented with information from Web-accessible documents and other grey literature from libraries and archives.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>High Quality Genomic DNA Extraction from Fungal Samples </p>

[GUIDELINES]
<p><strong>INTRODUCTION</strong></p>
<p>The OmniPrep™ for Fungus kit isolates high quality genomic DNA from fungal samples. The kit isolates high purity (A260/A280 ratios of 1.7 to 2) DNA between 100‐200kbp and the yield is 0.2‐1µg/5mg fungal samples. If used according to the protocols this kit purifies DNA from 1‐2gm fungal tissues.  </p>
<p> </p>
<p><strong>ITEM(S) SUPPLIED (Cat. # 786‐399)</strong></p>
<table>
<tbody>
<tr style="height: 20px;">
<td style="height: 20px;"><strong>Description</strong></td>
<td style="height: 20px;"><strong>Size</strong></td>
</tr>
<tr style="height: 20px;">
<td style="height: 20px;">Genomic Lysis Buffer</td>
<td style="height: 20px;">100ml</td>
</tr>
<tr style="height: 20px;">
<td style="height: 20px;">DNA Stripping Solution</td>
<td style="height: 20px;">10ml</td>
</tr>
<tr style="height: 20px;">
<td style="height: 20px;">Precipitation Solution</td>
<td style="height: 20px;">30ml</td>
</tr>
<tr style="height: 20px;">
<td style="height: 20px;">Longlife™ RNase (5mg/ml; &gt;60U/mg)</td>
<td style="height: 20px;">0.5ml</td>
</tr>
<tr style="height: 20px;">
<td style="height: 20px;">LongLife™ Proteinase K (5mg/ml)</td>
<td style="height: 20px;">2 x 0.5 ml</td>
</tr>
<tr style="height: 20px;">
<td style="height: 20px;">Molecular Grinding Resin™</td>
<td style="height: 20px;">1ml</td>
</tr>
<tr style="height: 20px;">
<td style="height: 20px;">TE Buffer</td>
<td style="height: 20px;">20ml </td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<p><strong>STORAGE CONDITIONS</strong></p>
<p>The kit is shipped at ambient temperature. Upon arrival, store the kit components as recommended on the label.  </p>
<p> </p>
<p><strong>ADDITIONAL ITEMS REQUIRED</strong></p>
<p>Chloroform, Isopropanol and 70% Ethanol  </p>
<p> </p>
<p> </p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Genomic  Signature Pattern Analysis of the HIV-1 Through Computational Approach
AUTHORS: Sushanta Kumar  Barik

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify">).</div><style>
							  .justify:after {
							    content: "";
							    display:inline-block;
							    width: 100%;
							  }
							</style></div></div>

[STEPS]
?. 
?. 
?. 
?. 
?. 5
?. 6
?. 7
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><span style="font-weight: 400;">Here we describe a procedure to make bacterial communication assays based on</span> 3OC6HSL and 3OC12HSL quorum sensing signaling. Strains that sense and response to these signals are plated on a permeable membrane printed with a hydrophobic grid, which is placed on top of an agar plate containing growth media and antibiotics<span style="font-weight: 400;">. This method is based on Grant et al. 2016 (</span>Grant PK, Dalchau N, Brown JR, Federici F, Rudge TJ, Yordanov B, Patange O, Phillips A, Haseloff J.Orthogonal Intercellular Signaling for Programmed Spatial Behavior. Mol Syst Biol. 2016 Jan 25;12(1):849. doi: 10.15252/msb.20156590)</p>
<p> </p>
<p><span style="font-weight: 400;">This assay has been used for low cost and open source fluorescence imaging (please see https://osf.io/dy6p2/ for further information &amp; data, https://github.com/SynBioUC/FluoPi for code and http://docubricks.com/viewer.jsp? for hardware assembly)</span></p>

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

[DESCRIPTION]
<p>Modified from Benjamin Schwessinger: High quality DNA from Fungi for long read sequencing e.g. PacBio. protocols.io</p>
<p>Optimized for DNA extraction from wheat stripe rust spores.</p>
<p>Buffers are best when fresh and not older than 3-6 months. <br />Buffered Phenol:Chloroform:Isoamylalcohol (25:24:1) should not be older than 3 months.</p>
<p>Critical steps to obtain high quality DNA:</p>
<ul>
<li>Do NOT heat samples during DNA extractions! Perform all steps at RT or 4oC as indicated.</li>
<li>Do NOT incubate samples with KAc for prolonged time periods</li>
</ul>
<p> </p>

[GUIDELINES]
<p>Modified from Benjamin Schwessinger: High quality DNA from Fungi for long read sequencing e.g. PacBio. protocols.io<br /><br />I am grateful for critical suggestion from Benjamin Schwessinger.<br /><br /></p>
<p>Optimized for DNA extraction from wheat stripe rust spores.</p>
<p><strong>Buffers are best when fresh and not older than 3-6 months. Buffered Phenol:Chloroform:Isoamylalcohol (25:24:1) should not be older than 3 months.</strong></p>
<p> </p>
<p>Critical steps to obtain high quality DNA:</p>
<ul>
<li>Do NOT heat samples during DNA extractions! Perform all steps at RT or 4oC as indicated.</li>
<li>Do NOT incubate samples with KAc for prolonged time periods</li>
</ul>
<p> </p>
<p><strong>Reagents required</strong></p>
<p><strong>BUFFER A:</strong> 0.35 M sorbitol</p>
<p>0.1 M Tris-HCl, pH 9</p>
<p>5 mM EDTA, pH 8</p>
<p>autoclave to sterilize</p>
<p> </p>
<p><strong>BUFFER B:</strong> 0.2 M Tris-HCl, pH 9</p>
<p>50 mM EDTA, pH 8</p>
<p>2 M NaCl</p>
<p>2% CTAB</p>
<p>autoclave to sterilize</p>
<p> </p>
<p><strong>BUFFER C:</strong> 5% Sarkosyl (N-lauroylsarcosine sodium salt SIGMA L5125)</p>
<p>Filter-sterilize</p>
<p> </p>
<p><strong>Other solutions</strong>:</p>
<p>Potassium Acetate 5M (KAc precipitate polysaccharides) pH 7.5</p>
<p>Polyvinylpyrrolidone (40000 MW) 1 % [w/v] (Sigma PVP40)</p>
<p>Filter-sterilize</p>
<p> </p>
<p>Isopropanol 100%</p>
<p>Ethanol 70%</p>
<p>Buffered Phenol:Chloroform:Isoamylalcool P:C:I (25:24:1, Sigma P2069)</p>
<p>Metal beads 5mm in diameter</p>
<p> </p>
<p><strong>Enzymes</strong></p>
<p>RNAse T1 (1000 U/ml, Thermo Fisher EN0541)</p>
<p><br />Lysis Buffer for 500 ul for 10-20 mg starting material</p>
<p>2.5 volume of Buffer A 187.5 ul</p>
<p>2.5 volume of Buffer B 187.5 ul</p>
<p>1.0 volume of Buffer C 75 ul</p>
<p>PVP 0.1 % 50 ul</p>
<p> </p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: pH-Rodo pathogens to monitor phagocytosis in pMacs
AUTHORS: Rebecca Wallings

[DESCRIPTION]
pH-Rodo pathogens to monitor phagocytosis in pMacs

[STEPS]
1. Once pMacs have adhered to plate, resuspend pHrodo‱ Green E. coli BioParticles‱ in Invitrogen‱ Live Cell Imaging Solution to a final concentration of 1mg/mL and sonicate in bath sonicator for 10 minutes. 

Add 100 μL of E. coli BioParticles solution to each well (96-well plate). 

Place cells in a live imaging chamber (37°C, 5% carbon dioxide, and 95% humidity) of an EVOS‱ M7000 (Invitrogen) at 20 x magnification capture 4 images per well every 20 minutes for 5 hours. 

Use brightfield images for automated-focus throughout the time course and use for normalization of GFP E. coli images.
TITLE: UC Davis - Creatinine Protocol
AUTHORS: Peter Havel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:  </span></div><div class = "text-block">The method employed here is based on an enzymatic colorimetric determination of creatinine which largely eliminates interferences known to the Jaffe method. In the final reaction sequence, the formation of the quinoneimine dye product results in an increase in absorbance at 550 nm (530-570 nm) which is directly proportional to the creatinine concentration in the sample. Potential interference from endogenous creatine and sarcosine are eliminated by the reaction of creatine amidinohydrolase, sarcosine oxidase and catalase before creatinine is determined. Ascorbate oxidase is included in the reagent to eliminate the influence of ascorbate in the sample.</div></div>

[STEPS]
?. Add 3 µl of calibrator and sample to each well.
?. Add 135 µl of R1 to each well. Incubate at 37°C for 5 minutes. Read at 550 nm.
?. Add 45 µl of R2 to each well. Incubate at 37°C for 5 minutes. Read at 550 nm.
?. Subtract blank readings from final readings. The assay will be linear so the unknown samples can be calculated as (Sample Absorbance ÷ Calibrator Absorbance) × Calibrator Concentration.
TITLE: Predation assessment on fake caterpillars and leaf sampling: Protocol for partner schools
AUTHORS: Bastien Castagneyrol, Elena Valdés-Correcher, Michèle Kaennel Dobbertin, Martin Gossner

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The '</span><span style = "font-style:italic;">Oak bodyguards</span><span>' citizen science project aimed to assess the effects of climate on two key biotic interactions occurring widely in natural and anthropized ecosystems, i.e., the top-down and bottom-up forces controlling  insect herbivory on leaves of the English oak (</span><span style = "font-style:italic;">Quercus robur</span><span>) trees. We chose the English oak as a model species as it is one of the most common and most emblematic forest trees in Europe, with a geographic range spanning more than 19 degrees of latitude. It is also widespread in both rural, suburban and urban environments. We exposed dummy plasticine caterpillars in trees to estimate predation rates. We additionally estimated leaf insect herbivory as the percentage of leaf area removed or damaged by insect herbivores. The </span><span style = "font-style:italic;">Oak bodyguards </span><span>project involved professional scientists and school children throughout Europe. We designed a simple protocol that was applied by both partner types. The protocol was written by scientists in collaboration with science instructors and communication officers. It was available in five languages: French, English, German, Spanish and Portuguese. The present document is the English version of the protocol. It describes how to estimate insect herbivory on oak leaves as well as predation rate on artificial prey made with green modelling clay. </span></div></div>

[STEPS]
TITLE: GST Bead pulldown Assay
AUTHORS: Adam  Yokom

[DESCRIPTION]
GST Pulldown Assay for recruitment of bait proteins to GST labeled prey proteins. Prey proteins can be purified or from lysate.

[STEPS]
2. Equilibrate 30uL of Glutathione Sepharose beads (GE Healthcare) into pulldown buffer. To do this, pipette 60uL of 50% slurry into a 1.7uL eppy. Add >500mL of wash buffer. Slow spin to pellet resin. ~1000rpm for 1 minute should be good. Repeat X3
4. Add 1-10 μM purified protein (add buffer of 25 mM HEPES pH 7.5, 150mM NaCl, 1mM MgCl2, 1mM TCEP to final volume of 200 uL). Alternatively, add 10mL of HEK293GnTi lysate.  
5. Let rock at 4C overnight. Wash x3 with buffer: 25 mM HEPES pH 7.5, 150mM NaCl, 1mM MgCl2, 1mM TCEP
7. Mix 17 uL eluent with 3 uL SDS-Loading dye. Heat samples for 5min @ 60C.
8. Run beads on SDS-PAGE gel and stain with Coomassie. 
1. Homogenize cell pellet with GST tagged protein in 0.5 ml of lysis buffer/protease inhibitors/1% TritonX-100. Clarify lysate by centrifugation at 40,000g for 15 min
3. Mix clarified lysate and washed GST resin together.
6. Elute washed resin with 50uL buffer: 25 mM HEPES pH 7.5, 150mM NaCl, 1mM MgCl2, 1mM TCEP + 25 mM glutathione
TITLE: E. coli and B. subtilis Colony PCR
AUTHORS: iGEM Dusseldorf

[STEPS]
?. Prepare a PCR master mix for 9 reactions, as follows:  ABC11x Reaction   (Volume; µL)9x Reactions   (Volume; µL)2Sterile MilliQ Water8723Red Taq 2x Master Mix(1.5 mM MgCl2)10904F Primer (10 µM)195R Primer (10 µM)196Total   Volume20180
ABC11x Reaction   (Volume; µL)9x Reactions   (Volume; µL)2Sterile MilliQ Water8723Red Taq 2x Master Mix(1.5 mM MgCl2)10904F Primer (10 µM)195R Primer (10 µM)196Total   Volume20180
?. Aliquot 20 µL of the master mix in 8 tubes of a PCR strip.
?. Transfer cells from a single colony (8 in total) using a sterile P2 pipette tip or inoculating loop
?. Run thermocycler using the following program (extension for 1.5 kb insert):  ABCD1StepTemp (°C)Time (mm:ss)Purpose21953:00Initial Denaturation325 cycles of Steps 2 to   4:42950:20Denaturation53570:30Annealing64721:00Extension75725:00Final Extension864∞Storage
ABCD1StepTemp (°C)Time (mm:ss)Purpose21953:00Initial Denaturation325 cycles of Steps 2 to   4:42950:20Denaturation53570:30Annealing64721:00Extension75725:00Final Extension864∞Storage
Extension time may vary depending on insert size
?. Run samples on a gel at 80-150 V until the dye line is approximately 75-80% of the way down the gel. A typical run time is about 1-1.5 hours, depending on the gel concentration and voltage.
TITLE: ATP assay
AUTHORS: Kiichi Hirota, Yoshiyuki Matsuo

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

[STEPS]
?. Cells were seeded in 96-well plates and allowed to grow for indicated time periods.
?. CellTiter-Glo reagent (50 μl) was then added directly into each well and incubated for 10 min prior to reading the plate using an EnSpire® Multimode Plate Reader (PerkinElmer, Waltham, MA, USA).This detected the luminescence generated by the luciferase-catalyzed reaction between luciferin and ATP.
?. The ATP content was then calculated by comparing the luminescence levels of cells with those of control samples, with the latter defined as 100%.Assays were performed in triplicate at least twice.
TITLE: Zebrafish larvae dissociation for FACs sorting cells expressing fluorescent proteins
AUTHORS: Abigail Elliot, Yi Feng

[DESCRIPTION]
Large number of transgenic lines with cell/tissue specific expressing of fluorescent proteins are available in the zebrafish field. These tools provide an opportunity for isolating cell tpe of interest for gene expression analysis. Key to success for a FAC sorting experiment is to generate single cells suspension with maximum number of viable target cells preserved. Here we describe the protocol that we optimized in the lab for generate single cell suspension for FAC sorting cells expressing fluorescent proteins from zebrafish larvae. We used a combination of enzyme digestion ( collagenase IV) and mechanical dissociation ( rapid pipetting) to generate single cell suspension, which achieved the highest number of cell recovery and viable cells. We have used this protocol for larvae at 3, 4, 5 dpf and for downstream single cell RNA sequencing and RNA sequencing analysis. 

[STEPS]
SECTION: Embryo Dissociation
1. Collect anaesthetised larvae in 2ml Eppendorf 5 min 
 


SECTION: Embryo Dissociation
2. Remove water and replace with 2ml Dissociation Solution with plastic pipette 5 min 

SECTION: Embryo Dissociation
3. Transfer larvae and solution to well of 12-well plate with plastic pipette. 2 min 

SECTION: Embryo Dissociation
4. Incubate on a shaker.    

SECTION: Embryo Dissociation
5. Vigorously pipette up and down with P1000, 30s each well. 30 s   

 

SECTION: Embryo Dissociation
6. Transfer digest to pre-cooled 2ml Eppendorf on ice – if pooling multiple tubes, do so at this step into a 25ml falcon tube.
SECTION: FACs preparation
7. Centrifuge collected tubes. 300-500 x g, 5 min, 4 °C 

 

SECTION: FACs preparation
8. Resuspend in Media A, 500ul / 50 larvae 
SECTION: FACs preparation
9. Filter through 40um filter, into FACS tube
SECTION: FACs preparation
10. FACs collect cells in collect live cells in Media A + 10% Goat Serum