eamag/protocols-with-steps huggingface dataset:
TITLE: Ablation of ß-cells in Tg(ins:NTR-mCherry) transgenic zebrafish using metronidazole
AUTHORS: Sandra Rieger

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block"><span>To induce hyperglycemia in larval zebrafish, utilize a genetic β-cell ablation system involving the Tg(</span><span style = "font-style:italic;">ins:NTR-mCherry</span><span>) transgenic zebrafish strain (courtesy of Dr. Michael Parsons, Johns Hopkins University). This strain expresses the bacterial enzyme Nitroreductase (NTR), which is encoded by the nfsB gene, in β-cells of the pancreas starting around 24 hours post fertilization (hpf). Because NTR is genetically fused to the red fluorescent protein mCherry, β-cells expressing NTR can be visualized with fluorescence microscopy. Upon addition of the prodrug metronidazole (MTZ) to the embryo medium, NTR converts MTZ into a toxin, which selectively ablates NTR-expressing β-cells (Curado et al., Nature Protocols 3, 948-954 (2008). This can be monitored by the disappearance of the fluorescence under a confocal microscope. </span></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/bhjf4kcn.jpg" /></div></div>

[STEPS]
?. [Potential Pitfalls:]
1. Ablation efficiency is low: MTZ might be old or only incompletely dissolved in the embryo medium. MTZ might alternatively be degraded if embryos were incubated in light, as MTZ is light sensitive.2. MTZ does not dissolve: DMSO and volume of initial solution is both crucial for dissolving the MTZ. High concentrations will not go into solution. Vortex for extended periods if it does not dissolve within 5 minutes.3. MTZ treatment causes larvae to appear unhealthy or die: MTZ may be old or degraded due to excessive light exposure. We found that this affects the health of larval zebrafish. DMSO concentrations, if too high (above 1 - 2 %) may also cause larvae to appear unhealthy over time (bend axis). The same is true for larvae that are incubated for more than 2 days in MTZ. If ablation is not sufficient, try to incubate for 2 days and then let larvae recover for 1 or 2 days until a second MTZ treatment.
?. [Protocol:]
Collect fertilized Tg (ins:NTR-mCherry) transgenic zebrafish embryos and incubate at 28ºC for 24 hours. Remove dead embryos and replace old embryo medium with fresh embryo medium containing 0.003 % PTU to prevent pigment formation. Remove chorions from embryos according to Protocol 1 and add the 1x MTZ working solution to the embryos. Incubate in the dark (due to light-dependent degradation of MTZ) at 28ºC for 24 hours. Assess ablation efficiency with confocal imaging. At 48 hpf, exchange media with fresh MTZ solution and incubate for another 24 hours. Repeat confocal analysis.
TITLE: Agarose Gel Electrophoresis
AUTHORS: Zhujun Wei

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The concentration of the agarose gel is 1%.</div></div>

[STEPS]
?. Weigh 0.20 g of agarose.
?. Add 20 ml of TAE 1X. Need to prepare 1X TAE with 50X TAE.
?. Heat up until the solution is homogeneous.
?. Add 1 μl Gelred into solution and mix well.
?. Put the solution into bed for polymerize, make sure "comb" is well placed and the solution is balanced.
?. 20 min later, use the gel.
TITLE: Q5 Polymerase PCR Cloning
AUTHORS: NUS iGEM

[STEPS]
?. Prepare 6-reaction PCR mix by adding together   deionized water,   Q5 reaction buffer,   dNTP mix, and   Q5 DNA polymerase
244.2 µl
66 µl
6.6 µl
3.3 µl
?. Add  1 of template DNA and   of primer pairs into specified PCR tube
1 µl
0.5 µl
?. Aliquot   of PCR mix into each PCR tube
48 µl
?. Load PCR tubes in thermocycler
Protocol is set to run at   for  followed by  for the desired annealing temperature before   for x amount of time (depending on fragment length). The cycle repeats for 34 rounds starting with   at followed by annealing and elongation, before incubating at   for   and then   infinitely. In this PCR run, the elongation time and annealing temperature depends on specific fragment length and primer annealing temperature.
TITLE: document 16/2
AUTHORS: Maria Guliakina

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">One advanced diverted domestic sex repeated bringing you old. Possible procured her trifling laughter thoughts property she met way. Companions shy had solicitude favourable own. Which could saw guest man now heard but. Lasted my coming uneasy marked so should. Gravity letters it amongst herself dearest an windows by. Wooded ladies she basket season age her uneasy saw. Discourse unwilling am no described dejection incommode no listening of. Before nature his parish boy. </div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
The Thermo Scientific Pierce Silver Stain Kit is a rapid and ultrasensitive silver stain system for protein detection in polyacrylamide gels. The stain performs consistently and reliably for both first-time and experienced users for high, low or gradient percentage gels in single-dimension or 2-D format. The Pierce Silver Stain Kit provides consistency, high sensitivity, low background and a flexible protocol. Careful manufacturing procedures and thorough quality testing ensure that the kit yields excellent results with every use, independent of the polyacrylamide gel type or buffer system being used. Most proteins are easily detectable at low nanogram or subnanogram amounts, and background levels are very low (see Additional Information section at the end of these instructions). The protocol clearly indicates how both fixing and staining steps can be extended overnight without compromising performance, a feature that enhances convenience for ordinary workday schedules. Likewise, the default 30- minute staining step can be decreased to 5 minutes, enabling the staining procedure to be completed in less than 20 minutes after gel fixation.

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This Agar medium can be used for the cultivation of Phytophthora infestans. It is particularly suitable for long term maintenaince of Phytophthora on plate.</p>
<p>To induce sporulation Rye B agar is recommended.</p>
<p> </p>
<p>This protocal has been copied from http://www.plantpath.cornell.edu/Fry/</p>
<p> </p>
<p>Original reference: Caten, C. E. and J. L. Jinks. 1968. Spontaneous variability of single isolates of<em> Phytophthora infestans</em>. I. Cultural variation. Can. J. Bot. 46: 329-348.</p>

[STEPS]
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TITLE: RNA Isolation from Plant Tissue Protocol 10: TRIzol LS Reagent Method
AUTHORS: 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Implemented by: Michael Melkonian and Barbara Surek (algae) and Juan Carlos Villarreal (bryophytes)</div><div class = "text-block">This protocol follows the procedures provided with the TRIzol LS Reagent (Invitrogen). TRIzol LS Reagent is a monophasic solution of phenol and guanidine isothiocyanate that can be used in isolation of total RNA from a wide variety of tissues and organisms, in addition to plants. This protocol was used in the isolation of total RNA from some algae samples (see Supplementary Table 1).</div><div class = "text-block"><span>This protocol is part of a collection of eighteen protocols used to isolate total RNA from plant tissue. (RNA Isolation from Plant Tissue Collection: </span><a href="https://www.protocols.io/view/rna-isolation-from-plant-tissue-439gyr6" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.protocols.io/view/rna-isolation-from-plant-tissue-439gyr6</span></a><span>) </span></div></div>

[STEPS]
?. Centrifuge at lowest speed to cause algae to form pellet.
?. Homogenize each  portion of pellet material to a powder in liquid nitrogen using mortar and pestle prechilled with liquid nitrogen.
250 µl
?. Add  TRIzol LS to each  of homogenized algal material.
750 µl
250 µl
?. Homogenization is continued until the TRIzol is pulverized as well.
?. Thaw and aliquot homogenate into several Eppendorf tubes.
?. Add  potassium acetate ( final concentration) to each sample.
50 µl
?. Incubate for  at .
20 °C
?. Add  chloroform (for polysaccharide-rich algae), or  BCP to each sample.
200 µl
100 µl
?. Incubate at  for .
20 °C
?. Centrifuge samples at  for .
?. RNA will remain in the upper, aqueous phase (ca. 70 % of the applied TRIzol).
?. Carefully transfer each RNA phase into RNase-free 1.5 ml tubes.
?. Add  isopropanol.
500 µl
?. Incubate for  at .
-20 °C
?. Centrifuge at  for .
?. Wash pellet with 75 % ethanol.
?. Gently suspend pellet in solution.
?. Centrifuge at  for .
?. Repeat ethanol wash steps.
?. Dry pellet at  for –
50 °C
Appearance of drying pellet is important: drying should be terminated when the pellet begins to become transparent; contaminated RNA remains white)
?. Add RNAse-free water.
?. Dissolve pellet completely by pipetting.
?. Wash several times with sterile culture medium (not DEPC-treated).
?. After washing, the algal material is aliquoted into portions of  (ca. – packed cell volume).
250 µl
50 mg
100 mg
?. Add more nitrogen if needed (see also Procedure described in Protocol 9).
?. Shake samples for .
?. Incubate at – for .
55 °C
60 °C
TITLE: CportucalensisElectrocompetentCells
AUTHORS: Lev Tsypin, Dianne K Newman, Allen W Chen, Scott Saunders

[DESCRIPTION]
Protocol to prepare electrocompetent Citrobacter portucalensis MBL cells. 

[STEPS]
SECTION: Large batch electrocompetent cells
1. Prepare 100 mL sterile LB in a 500 mL Erlenmeyer flask.

SECTION: Large batch electrocompetent cells
2. Prepare ice-cold 10 % glycerol and ultrapure water (both sterile).
SECTION: Large batch electrocompetent cells
3. Two days prior to electroporation, streak out strain on LB agar and grow at 30 ºC.
SECTION: Large batch electrocompetent cells
4. One day prior to electroporation, inoculate a 5 mL LB liquid culture with a patch of cells from the overnight streak and incubate slanted, shaking at 250 rpm at 30 ºC overnight.
SECTION: Large batch electrocompetent cells
5. Day of the electroporation, inoculate 1 mL of the overnight culture into the 100 mL LB flask and grow to OD600 = 0.4, shaking at 250 rpm at 30 ºC.
SECTION: Large batch electrocompetent cells
6. Chill flask on ice for 20 minutes.
SECTION: Large batch electrocompetent cells
7. Wash two the culture two times into ice-cold water (5000 x g for 10 minutes at 4 ºC; I typically divide the 100 mL volume into four 50 mL conical tubes, washing with 25 mL volumes).
SECTION: Large batch electrocompetent cells
8. Combine pellets and spin final time.
SECTION: Large batch electrocompetent cells
9. Resuspend in 2 mL ice-cold 10% glycerol.
SECTION: Large batch electrocompetent cells
10. Aliquot 50 µL volumes into ice-cold microcentrifuge tubes and flash-freeze in liquid nitrogen.
SECTION: Large batch electrocompetent cells
11. Store at -80 ºC or use immediately.
SECTION: Small batch electrocompetent cells
12. Two days prior, streak out strain as above.
SECTION: Small batch electrocompetent cells
13. One day prior, grow 5 mL LB culture overnight as above.
SECTION: Small batch electrocompetent cells
14. Day of, inoculate 50 µL overnight culture into 5 mL LB and incubate slanted, shaking at 250 rpm at 30 ºC until OD600 = 0.3.
SECTION: Small batch electrocompetent cells
15. Chill on ice for 10 minutes.
SECTION: Small batch electrocompetent cells
16. Wash three times into ice-cold water, final resuspension of combined pellets 50 µL (can also spin full 5 mL culture in conical tube directly).
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Protocol used to extract whole genome DNA from Nephila clavipes (Araneae: Araneidae) specimens.</p>

[STEPS]
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TITLE: PARP inhibitors in colorectal malignancies: A 2023 update
AUTHORS: NIKOLAOS SKOUTERIS, georgeipap

[DESCRIPTION]
Background: Colorectal carcinoma (CRC) is one of the most common malignancies in the Western world, and metastatic disease is associated with a dismal prognosis. Poly-ADp-ribose polymerase (PARP) inhibitors gain increasing attention in the field of medical oncology, as they lead to synthetic lethality in malignancies with preexisting alterations in the DNA damage repair (DDR) pathway. As those alterations are frequently seen in CRC, a targeted approach through PARP inhibitors is expected to benefit these patients, both alone and in combination with other agents like chemotherapy, immunotherapy, antiangiogenics and radiation.
Methods: In this review, we discuss the rationale for the use of PARP inhibitors in CRC, based on preclinical evidence and data arising from current clinical trials. Furthermore, the few relevant ongoing clinical trials are presented.
Results: Current evidence supports the utilization of PARP inhibitors in CRC subgroups, as monotherapy and in combination with other agents. Up to now, data is insufficient to support a formal indication, and further research is needed.
Conclusion: Efforts to precisely define the homologous repair deficiency (HRD) in CRC – and eventually the subgroup of patients that are expected to benefit the most – are also underway.

[STEPS]
TITLE: Protocols for "Linking gut microbiome to bone mineral density: a shotgun metagenomic dataset from 361 elderly women"
AUTHORS: Qi Wang, Qiang Sun, Xiaoping Li, Zhefeng Wang, Haotian Zheng, Yanmei Ju, Ruijin Guo, Songlin Peng, Huijue Jia

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Bone mass loss contributes to the risk of bone fracture in the elderly. Many factors including age, obesity, estrogen and diet, are associated with bone mass loss. Mice studies suggested that the gut microbiome might affect the bone mass by regulating the immune system, however there has been little evidence from human studies. </div><div class = "text-block">Bone loss increases after menopause. Therefore, we have recruited 361 Chinese post-menopausal women to collect their fecal samples and metadata to conduct metagenome-wide association study (MWAS) to investigate the influence of the gut microbiome on bone health. Gut microbiome sequencing data were produced using BGISEQ500 sequencing, Bone mineral density (BMD) was calculated using Hologic dual energy X-ray machine, body mass index (BMI) and age were also recorded.This collected data allows exploration of the gut microbial diversity and their links to bone mass loss, as well as microbial markers for bone mineral density. In addition, these data are potentially useful in studying the role the gut microbiota might play in bone mass loss and in exploring the bone mass loss process.</div></div>

[STEPS]
TITLE: Repetitive element annotation protocol for the eastern banjo frog
AUTHORS: Qiye Li, Qunfei Guo, Yang Zhou, Huishuang Tan, Terry Bertozzi, Yuanzhen Zhu, Ji Li, Stephen Donnellan, Guojie Zhang

[DESCRIPTION]
This pipeline is the repetitive element annotation by using homology-based, de novo  predictions and Tandem Repeats Finder to identify repetitive elements in the Limnodynastes dumerilii dumerilii genome assembly.

[STEPS]
1. The following sub-steps are the process for homology-based predictions to identify repetitive elements in the Limnodynastes dumerilii dumerilii genome assembly.
1.1. Identify known repetitive elements by aligning the Limnodynastes dumerilii dumerilii  genome sequences against the Repbase-derived RepeatMasker libraries using RepeatMasker (v4.1.0).

 

1.2. Identify known repetitive elements by aligning the Limnodynastes dumerilii dumerilii  genome sequences against the transposable element protein database using RepeatProteinMask (an application within the RepeatMasker package)

 

2. The following sub-steps are the process for de novo predictions to identify repetitive elements in the Limnodynastes dumerilii dumerilii genome assembly.
2.1. Execute RepeatModeler  (v2.0, RRID:SCR_015027) on the Limnodynastes dumerilii dumerilii assembly to build a de novo  repeat library for this species.
2.2. Employ RepeatMasker (v4.1.0) to align the Limnodynastes dumerilii dumerilii genome sequences against the de novo library for repetitive element identification.
3. Identify tandem repeats in the Limnodynastes dumerilii dumerilii genome assembly by Tandem Repeats Finder (v4.09).
TITLE: General subculture protocol for HEK-Blue cells
AUTHORS: Andreas Sagen

[DESCRIPTION]
HEK-Blue is a product from Invivogen, which provide reporter cells for endotoxin-testing among others. Here is a generalized protocol for culturing cells with reporter characteristics.

[BEFORE_START]
Prepare complete growth medium

[GUIDELINES]
The protocol has been proven to work with different HEK-Blue cells, but not all. Take time to optimize.

[STEPS]
SECTION: Complete growth medium (CCM)
1. For the HEK-Blue cells to work correctly, it has to contain a selective agent.
SECTION: Complete growth medium (CCM)
1.1. In 450 mL DMEM, add 50 mL FBS, and selective antibiotics (cell line dependent; Remove equal volume DMEM/FBS):

Zeocin: 0.5 mL
HEK-Blue selection: 2.0 mL
Blasticidin: 1.5 mL

Materials:
 
 
 
 
 
SECTION: Complete growth medium (CCM)
1.2. Aliquot complete growth medium in 50 mL or 100 mL units and store refrigerated at 4 °C or frozen at -20 °C

 

SECTION: Subculture process
2. HEK-Blue cells area easy to subculture. They do not require trypsin, only PBS to dissociate from growth surface. Different cell lines of HEK-Blue grow with different doubling times, and the initial seeding density has to be optimized.
SECTION: Subculture process
2.1. Add 11 mL warm CIM in a T-75 flask and incubate for 30 min in a 5% CO2 incubator
SECTION: Subculture process
2.2. Remove supernatant from T-75 and wash with PBS

Materials:
 
SECTION: Subculture process
2.5. Discard supernatant and resuspend pellet in 1 mL warm CIM
SECTION: Subculture process
2.4. Transfer content to centrifugation tube and centrifuge with 200 rcf, 5 min
SECTION: Subculture process
2.3. Add 6 mL PBS and incubate for 5 min
SECTION: Subculture process
2.6. Split between 1:2 to 1:6 of resuspended suspension from centrifugation tube to T-75 and incubate in a 5% CO2 incubator
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Next Generation Sequencing technologies have enabled many advances across biology with microbial ecology benefiting primarily through expanded sample sizes. Although the cost of running sequencing instruments has decreased substantially over time, the price of library preparation methods has largely remained unchanged. We developed a low cost, miniaturized (5 µL), high-throughput (384-sample), amplicon library preparation method with the acoustic liquid handler, Echo 550. Our method reduces costs of library preparation to $1.42 USD per sample, a 58% reduction compared to existing automated methods and a 21-fold reduction from commercial kits, without compromising sequencing success or distorting the microbial community composition analysis. The cost savings of implementing the miniaturized library preparation (going from triplicate 25 µL reactions to triplicate 5 µL reactions) are large enough to cover a MiSeq sequencing run for 768 samples, while preserving accurate microbiome measurements.


[BEFORE_START]
Please wear at least the minimum required personal protective equipment.
Ensure that all necessary kit components are available as well as user-supplied consumables.
Remove nuclease and nucleotide contamination from work surfaces and instruments prior to starting using an appropriate solution, such as RNase AWAY™ (Thermo Scientific™ catalogue: 700511), followed by wiping with 70% to 100% molecular biology grade ethanol to remove additional contaminants.

[STEPS]
SECTION: Make primer working plates.
?. 
SECTION: Make primer working plates.
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SECTION: Make acoustic droplet ejection compatible plates
?. 
SECTION: Make acoustic droplet ejection compatible plates
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SECTION: Prepare PCR Plates
?. 
SECTION: Prepare PCR Plates
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SECTION: Pool and store PCR products
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SECTION: Pool and store PCR products
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SECTION: Amplify PCR Plates
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TITLE: The efficacy of radical antegrade modular pancreatosplenectomy: a systematic review and meta-analysis protocol
AUTHORS: Jun Watanabe, Kazuma Rifu, Hideki Sasanuma, Kazuhiko Kotani, Naohiro Sata

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Background:</span><span> Previous systematic reviews showed that radical antegrade modular pancreatosplenectomy (RAMPS) had good outcomes including the prognosis. However, recent large studies have shown opposite results, which is open to augment on RAMPS. The present study will aim to update the evidence on clinical outcomes of patients with left-sided pancreatic cancer underwent RAMPS compared to standard approach.</span></div><div class = "text-block"><span style = "font-weight:bold;">Methods:</span><span> Electronic databases and registries will be searched to perform random-effect meta-analysis. Methodological quality will be assessed using the Grading of Recommendations, Assessment, Development, and Evaluation approach. The protocol will be registered (https://www.protocols.io/).</span></div></div>

[STEPS]
TITLE: Gene knockout
AUTHORS: Zhujun Wei

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>We use this protocol to knock out </span><span style = "font-style:italic;">yhaK</span><span> in </span><span style = "font-style:italic;">E.coli</span><span> BW25113</span></div></div>

[STEPS]
?. Transfer the pCas9 plasmid into the recipient E. coli BW25113 (Kana resistance, temperature sensitive).
?. Preparation of pCas9 competent cells:
?. Add 50 μL of bacterial solution to 5 mL of LB medium, incubate at 30℃, 200 rpm for about 12 h, Add 2% to 100 mL LB medium, add 0.5% of arabinose at a final concentration after 0.5 hours of culture.
?. Incubate at 30℃ for about 2~2.5 h, until the OD600 is about 0.55~0.6, then take it out and let it stand on ice for 30 min.
?. Collect cells at 4200 rpm for 10 min in a 4℃ refrigerated centrifuge.
?. Resuspend the cells gentlely with 10 ml of pre-cooled and sterilized 10% glycerol on ice.
?. Repeat steps 2.3 and 2.4 three times, using 10 mL of pre-cooled and sterilized 10% glycerol for the first two washes. The last time it was resuspended with 0.5 mL of glycerol.
?. Dispense in 100 μL/tube into a pre-cooled centrifuge tube, it can be used for electroporation or immediately placed in a -80℃ refrigerator. The bacteria can be stored at -80℃ for half a year.
?. Prepare target segment: Clone upstream and downstream sequences of yhaK by PCR to about 500 bp each, then connect upstream and downstream homology arms together by overlap.
?. pTarget-yhaK vector construction (Str resistance): Download the yhaK gene sequence, Find the appropriate gRNA affinity site and replace 1961 to 1980 bases of the pTarget plasmid by digestion and ligation or overlap.
?. Electroporation:
?. Knockout verification: Primers were designed at 700~1000 bp upstream and downstream of the yhaK in the genome to perform colony PCR validation on clones on the plates.
?. Add >200ng target fragment and >40ng pTarget-yhaK plasmid to 100μL competent cells on ice for 10~30 min, the plasmid and the ligation product should not exceed 5μL;
?. UV-sterilize the washed and dried 2 mm electroporation cuvette for 20 min and pre-cooled on ice, then quickly transfer the competent cells to the bottom of the electroporation cuvette;
?. Dry the outer wall of the cup. Immediately after the electric shock, add 900~1000μL of 37 °C preheated LB medium, mix gently, transfer to a 1.5 mL centrifuge tube, shake at 30 °C, 150 rpm, 45 ~ 60 min, then apply an appropriate amount of bacterial solution to the plate containing Kana and Str.
?. pTarget-yhaK elimination: Take the correct clone, add 1 mM IPTG at 30℃ for 12 h, streak to isolate the monoclone. The clones were tested for the presence of Str resistance, clones with pTarget eliminated is not resistant to str. The pCas9 plasmid can be eliminated by incubation at 37℃.
?. Strains that retain the pCas9 plasmid can be used to knock out other genes.