eamag/protocols-with-steps huggingface dataset:
TITLE: SPARC - Preparation of Plasma Samples from Rats
AUTHORS: J Paul Robinson

[DESCRIPTION]
Objective: To collect plasma from live rat experiments for storage at -20˚C for further hormone assays.

[STEPS]
1. Turn on the Hettich Mikro 22R refrigerated centrifuge so it can begin to cool down to 4 °C.
 
 
 

2. Add 15 µL of  Protease Inhibitor Cocktail to each of 5 Culex tubes (Vial Clear 8mm Crimp Rnd Bottom 
300 µl Lot:0000071223 MicroLiter Wheaton Company) 
 
Firmly place cap (Snap Cap 8mm, Natural with Cut T/S Septa Lot 0000000308 MicroLiter Wheaton Company) making sure it is on good and level. (these are plastic reusable labels from BASI inc)

3. Place the plastic numbers (re-usable-do not dispose of these number labels)1-5 one on each Culex tube, place up from bottom of tube.

 

4. Place the Culex tubes on the Culex machine in slots 1-5.
5. Put the labels on the strip tubes if they are not already.

 

6. When the first sample comes off the Culex, centrifuge on program #3 (4˚C, 10 minutes, 1000 x g (3.000 rpm)1000 x g  immediately.

7. Using the P100 pipette set at 85 µLl carefully try to remove as much of the plasma as possible and transfer to the appropriate pcr strip tube. If needed set to 15 µLand remove remaining plasma without disturbing the red cell layer.

 

8. If needed switch to the P100 set to 15 µL and remove remaining plasma without disturbing the red cell layer.

9. Keep the strip tubes in the freezer block (ISO Freeze) during the entire collection process.
 

10. Do this for each sample as it comes off the Culex machine, S1, S2, S3, S4, S5.
11. Place an orange dot on the label on top of all the tubes that have been collected using the orange sharpie. This just indicates the original tube from which the aliquots were taken.
12. Once the fifth sample has been collected, change the rotor on the Hettich Mikro 22R centrifuge to the one for strip tubes.
13. Change the program to program #2, which is 6.000 rpm (RCR 4020)10 minutes, 4˚C.

14. You will need to close the lid and press start and wait. It will recognize the rotor has been changed and will automatically STOP.
15. Once it is stopped open the lid again. Then close the lid and press START. It should now run through the program.
16. When stopped, remove the strip tube.
17. Using the P200 set to 25 µL and the P100 and make two aliquots of 25 µL for every sample. If any aliquot is short, write an “S” on the cap of that aliquot.

18. Once all samples have been aliquoted, spin them again on program #2 to get all the sample to the bottom.
19. 25 µL samples must be transported using the ISO Freeze sample rack to keep temperature at 4 °C

20. Make sure all lids are on tight, and place samples in -20 °C freezer. Samples are added over time to the next plate to be run and placed in a labeled Ziploc bag in the -20 °C freezer.

21. Samples must then be logged into the next plate map using the MPLEX software. Print a copy after each sample set has been added to the plate map for recording purposes. The purpose of adding each sample to the Plate Map for the MPLEX software is to maintain a fully digitized record for each assay.
TITLE: Jimmy Moore Keto Reviews 2022″ Side Effects, Best Results, Works &amp; Buy!
AUTHORS: jimmymooreketopills  

[DESCRIPTION]
Jimmy Moore Keto - The keto diet has gotten the standard all insanely standard in each of the a few years, and there's a major consideration or that. Today, we empower you about another update called Jimmy Moore KetoCapsule. This formula supports keto sister and assists clients with seeing the upheld results they can consistently. Keto might be that the most striking eating routine regimens out there, anyway like all weight control plans, will be a standard that is hard to begin or keep up with for clients.

[STEPS]
TITLE: U Mass - Triglyceride
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">This experiment involves a spectrophotometric measurement using Roche Cobas Clinical Chemistry Analyzer.  Triglyceride levels may be measured in serum, plasma, and tissue samples. Serum and tissue triglyceride levels are affected by alterations in systemic lipid metabolism, lipid digestion/absorption, and lipid clearance.  Serum and tissue triglyceride levels are altered in obesity, insulin resistance, type 2 diabetes, alcoholic steatohepatitis, non-alcoholic fatty liver disease, and non-alcoholic steatohepatitis.</div></div>

[STEPS]
?. Perform daily quality control assessment of instrumentation before analysis.
?. Load each sample into a specialized micro-sample cup for the clinical chemistry analyzer.
?. Select Triglyceride test on display and run the analysis.
?. Collect and analyze the data.
TITLE: The molecular neuropathology of amyotrophic lateral sclerosis: protocol for systematic review and meta-analysis
AUTHORS: Elizabeth Elliott, Jenna M Gregory, Arpan R Mehta, Siddharthan Chandran, Colin Smith.

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A significant body of neuropathological research examining the molecular heterogeneity across ALS phenotypes exists and an up to date formal review of these studies is now required. Through conducting a systematic review and meta-analysis of the existing ALS human neuropathology literature we aim to outline the current pathological molecular profile of ALS. </div><div class = "text-block">A search will be conducted without language or publication date restrictions across three databases; 1. PubMed, 2. MEDLINE, 3. EMBASE. Studies comparing human ALS or ALS-FTD brain and/or spinal cord samples to controls will be selected. The primary outcome measure will be molecular species quantification followed by an assessment of study heterogeneity as a secondary outcome measure. The Systematic Review Facility online screening tool (app.syrf.org.uk) will be used by two independent authors to screen the titles and abstracts based on predefined criteria. Data will be extracted by one reviewer and an assessment of study quality will be conducted against the CAMARADES’ study quality checklist (adapted). Additional study quality assessment and data extraction will be carried out by an independent reviewer for 10% of the selected studies. Where appropriate, standardised mean differences (SMD) will be used to describe outcome measures to enable inter-study comparison.</div><div class = "text-block">Advances in transcriptional profiling technology have generated a variety of high throughput platforms, ranging from microarray gene chips with a specific range of oligonucleotide probes through to sequencing at a whole genome level. Human pathological studies to date have revolutionised our understanding of the ALS disease aetiology. Here we aim to review the outcomes of these studies across the field as a whole and to consider the impact of methodological heterogeneity on the results presented. </div></div>

[STEPS]
TITLE: DNA Purification from a PCR Product (Protocol for NucleoSpin® PCR clean-up Gel Extraction Kit)
AUTHORS: Alba Balletbó

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The following protocol is suitable for PCR clean-up as well as DNA concentration and removal of salts, enzymes, etc. from enzymatic reactions (SDS < 0.1 %).</div></div>

[STEPS]
?. Adjust DNA binding condition, For very small sample volumes 'Note': For removal of small fragments like primer dimers dilutions of Buffer NTI can be used instead of 100 % Buffer NTI. Incubate the mixture at 50ºC for 5-10 minutes or until the gel has completely melted.
?. Place a NucleoSpin® PCR clean-up Gel column in a provided 2 mL collection tube. Apply up to 700 μL of the DNA/agarose solution to the NucleoSpin® PCR cleanup Gel column, and centrifuge at 11, 000 x g for 30 seconds at room temperature.
?. Discard liquid and place the NucleoSpin® PCR clean-up Gel column back into the same collection tube. For volumes greater than 700 µL, load the column and centrifuge successively, 700 μL at a time. Each NucleoSpin® PCR clean-up Gel column has a total capacity of 25 μg DNA. If the expected yield is larger, divide the sample into the appropriate number of columns.
?. Add 700 μL of Buffer NT3 into the NucleoSpin® PCR clean-up Gel columnCentrifuge at 11,000 x g for 30 seconds at room temperature to wash the column. Discard the flow-through and re-use the collection tube.
?. Repeat step 4 with another 700 μL of Buffer NT3.
?. Discard liquid and centrifuge the empty NucleoSpin® PCR clean-up Gel column for 1 minute at 11,000 x g to dry the column matrix. Do not skip this step, it is critical for the removal of ethanol from the NucleoSpin® PCR clean-up Gel column.'Note': Residual ethanol from Buffer NT3 might inhibit enzymatic reactions. Total removal of ethanol can be achieved by incubating the columns for 2–5 minutes at 70ºC prior to elution.
?. Place a NucleoSpin® PCR clean-up Gel column into a clean 1.5 mL eppendorf tube. Add 15-30 μL (depending on desired concentration of final product) of Elution Buffer (10 mM Tris-HCl, pH 8.5) directly onto the column matrix and incubate at room temperature for 1 minute. Centrifuge for 1 minute at 11,000 x g to elute DNA. This represents approximately 70% of bound DNA. An optional second elution will yield any residual DNA, though at a lower concentration.'Note': DNA recovery of larger fragments (> 1000 bp) can be increased by multiple elution steps with fresh buffer, heating to 70ºC and incubation for 5 minutes.
TITLE: 10x Protocols: Chromium Next GEM Single Cell 3ʹ -- University of Minnesota TMCs (CG000315 Rev E)
AUTHORS: Laura J Niedernhofer, David A Bernlohr

[DESCRIPTION]
Following single-cell dissociation or nuclei isolation, the Next GEM 3' assay uses microfluidics to partition and assign cell- or nuclei-specific barcodes to transcript cDNA at the 3' end. Barcoded cDNA is prepared with adaptors for sequencing by synthesis (SBS). The following protocol has been adapted from protocols developed by 10x Genomics and Illumina to be used at the University of Minnesota TMCs in collaboration with the University of Minnesota Genomics Center. These protocols are owned by their respective companies and are subject to periodic revision.

[STEPS]
SECTION: Library Preparation
2.   

 

 

SECTION: FASTQ Generation
3. BCL data from Illumina sequencer is demultiplexed and converted into FASTQ format using bcl2fastq version 2.20.0
SECTION: Tissue Preparation
1. Complete single cell or nuclei isolation prior to starting this protocol
TITLE: Fluorescent Immunolabelling for endogenous ChAT in mice striatum
AUTHORS: Roberta Marongiu

[DESCRIPTION]
This immunohistochemistry protocol has been designed and adjusted specifically for ChAT immunolabelling in the mice striatum 

[STEPS]
2. Wash:  3 x 10 min  gently on a shaker, in TBST at Room temperature .

4. Blocking step: Incubate the slides with blocking solution on the shaker for 60 min  at Room temperature  .
6. Primary antibody: incubate the slides with the blocking solution/Antibody on a shaker at 4 °C  for 1440 min  2880 min.

7. Washes: 3 x 10 min on a shaker, in TBST.

9. incubate the slides with Secondary antibody solution 60 min  at Room temperature.

10. Washes: 2 times, for 10 min   in TBST.

12.  Incubation the slides 10 min with the DAPI solution.

13. Washes: 2 times, for 10 min  each in TBST.

14. Mounting on Superfrost+ slides.
15. Coverslip: mounted with prolong anti-fade reagent (Invitrogen).
1. Select floating sections of mouse striatum (30um thick).
3. During the washes, prepare blocking solution: 10 % donkey serum (DS) in TBST.
5. Before the end of the blocking step, prepare the solution with the primary antibody ChAT
blocking solution (TBST+10% DS) + Goat Anti-Chat Ab  dilution 1:200.
8. During the washes, prepare the secondary Antibody solution
blocking solution (TBST+10% DS) + anti donkey anti goat Alexa Fluor 1:1000.
11. Optional: DAPI staining
During the washes, prepare the DAPI solution 
DAPI (1 mg/ml). Dilution 1:10.000 in TBST.
TITLE: IPTG
AUTHORS: Erçağ Pinçe

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Preparation of Inducer IPTG for expression of CFP and YFP </div></div>

[STEPS]
?. [Dissolving IPTG]
Dissolve 2.383 g of IPTG in 8 mL double distilled water(milliQ)
?. [Complete the volume of water to 10mL]
Add more ddH2O to bring the total volume to 10mL
?. [Filter sterilize ]
Filter sterilize with a 0.22 μ- syringe filter.
?. [Aliquot and store]
Store in 1mL aliquots at -20 °C.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes the recipe for a standard sugar-agar food source for fruit flies</p>

[GUIDELINES]
<p>This protocol describes how to make enough sugar-agar solution for approximatley 60 Drosophila storage vials (23ml; Sarstedt 58.490). </p>
<p>Once the sugar-agar food is decanted into vials, the vials should be left covered but not sealed, at room temperaure until the food has set and cooled. The vials can then be sealed with cotton wool/bung and stored at 4°C for approximately 4 weeks before use.</p>
<p>Food vials should be brought to room temperature before flies are added.</p>

[STEPS]
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TITLE: Tau aggregation monitored by thioflavin-T (ThT) fluorescence in a plate reader
AUTHORS: Patricia Yuste-Checa, F Ulrich Hartl

[DESCRIPTION]
This protocol details how to efficiently monitor Tau aggregation by thioflavin T fluorescence in a plate reader.

[STEPS]
SECTION: Tau aggregation monitored by thioflavin-T (ThT) fluorescence in a plate reader
1. Mix reagents to a final concentration of 10 micromolar (µM) Tau (TauRD), 2.5 micromolar (µM) Heparin, 2 millimolar (mM) MgCl2, 10 micromolar (µM) ThT in 1x PBS pH 7.2. Prepare a mix for 4.5 reactions per condition (per condition, four technical replicates in each plate). Final volume is 80 µL per well.
 

SECTION: Tau aggregation monitored by thioflavin-T (ThT) fluorescence in a plate reader
2. Dispense 80 µL of the mix into a well of a 96 well half-area plate of black polystyrene with a clear bottom (Corning 3881).

SECTION: Tau aggregation monitored by thioflavin-T (ThT) fluorescence in a plate reader
3. If possible, the outer wells should not be used and instead filled with water.
SECTION: Tau aggregation monitored by thioflavin-T (ThT) fluorescence in a plate reader
4. Seal the plate with parafilm to avoid evaporation.
SECTION: Tau aggregation monitored by thioflavin-T (ThT) fluorescence in a plate reader
5. Set the following parameters in a SPARK multimode microplate reader (TECAN) and start the reaction:
Fluorescence measurement: ThT signal, excitation 440 nm, emission 480 nm, (use gain regulation) measured every 2 minutes.
Temperature 37 °C
Constant shaking (50 seconds linear shaking: amplitude 4.5 mm, frequency 420 rpm - 50 seconds orbital shaking: amplitude 1.5 mm, frequency 360 rpm).
TITLE: Annealing Oligonucleotides (Instructor Protocol)
AUTHORS: Brian    Teague

[DESCRIPTION]
This is the instructor protocol for
 



[STEPS]
SECTION: Lab Setup
1. Prepare 15 ml conicals with 5 mL   in each. Prepare one tube for each 4 students (2 groups of 2).
SECTION: Instructor Tips &amp; Common Student Errors
3. Instructor Tips
This is the first "real" lab. I generally start the class explaining what annealing is and why we're doing it, point out where the materials are, and then sit down. My approach is deliberately hands-off.
Each semester, I am amazed that this protocol takes students an entire two-hour lab period. It probably has to do with my hands-off approach, as mentioned above.
Students often struggle with the dilution math. Remind them of c1 * v1 = c2 * v2 and remind them that they've already done this once. Suggest that they review the dilutions and pipetting lab and that they ask their colleagues for help. Only if students are really stuck will I review the procedure with them.
We've only got one thermocycler -- but this reaction is fine to wait until the entire class has loaded their samples to start. (As opposed to the PCR, which needs to stay on ice until it's time to start.) So I ask students to load their samples into the thermocycler as they finish.
As students load their samples, double-check that they look like about 10 µl of liquid. Significantly more may be a sign of a pipetting error.
As students load their samples, double-check that they're labeled. Remind them that their tube will be one of many and ask if they'll be able to pick their tube out from all the others?
Remind students to record their tube label in their notebook.
SECTION: Instructor Tips &amp; Common Student Errors
4. Common Student Errors
Difficulty doing the dilution math.
Didn't mix the sample well
Didn't label their tubes
SECTION: Lab Setup
2. Program a thermocycler with the following program:

Step 1: 95°C for two minutes
Step 2: 95°C for 45 seconds, decreasing one degree each cycle
Step 3: Go to step 2 70 times
Step 4: Store at 8°C
TITLE: Mitophagy experiments
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol describes how to induce mitophagy in HeLa cells.

[STEPS]
SECTION: Mitophagy experiments
1. To induce mitophagy, treat cells with 10 micromolar (µM) Oligomycin and 4 micromolar (µM) Antimycin A.
SECTION: Mitophagy experiments
2. In case cells are treated for more than 480 min, add 10 micromolar (µM) Q-VD-OPh to suppress apoptosis.
SECTION: Mitophagy experiments
3.  Analyze the samples by SDS-PAGE and western blot or flow cytometry.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<header>We analyzed the recently available whole genome sequences from two thraustochytrids (Aurantiochytrium limacinum ATCC MYA-1381, Schizochytrium aggregatum ATCC 28209) and one aplanochytrid (Aplanochytrium PBS07) We then calculated the genome-wide relative synonymous codon usage, codon frequencies and GC content for predicted coding sequences from each of the three species. We compared these to other stramenopiles: the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana, and the oomycete Phytophthora sojae, as well as to the ascomycete fungus Saccharomyces cerevisiae. See <a href="https://you.stonybrook.edu/labyrinthulomycetes/coding-properties/" target="_blank">this page </a>for further description.</header><header></header><header>This code was run in R version 3.3.2 (2016-10-31)</header><header>
<p><span style="font-weight: 400;">Package info: RCurl_1.95-4.8 bitops_1.0-6   ape_4.0        reshape_0.8.6  seqinr_3.3-3</span></p>
 </header>
<div> </div>

[STEPS]
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TITLE: Trace Metals Solution
AUTHORS: Adrien Assie, Buck Samuel

[DESCRIPTION]
Large quantities of C. elegans can be grown in a liquid medium. Liquid cultures of C. elegans are usually grown on S Medium using concentrated E. coliOP50 as a food source. This is the Trace Metals solution recipe to complement the S complete medium solution.

[GUIDELINES]
Store in dark or light protected bottle


[STEPS]
1. Start with700 mL

2. 10 mLof *500 mM EDTA, pH 8.0 or 1.86 gNa2EDTA.2H2O (5 mM)


3. 0.69 gFeSO4 ∙7H2O (2.5 mM)

4. 0.20 gMnCl2 ∙ 4H2O (1 mM)

5. 0.29 g ZnSO4 ∙7H2O (1 mM)

6. 0.016 gCuSO4 (0.1 mM)

7. Adjust to 1000 mL

8. Autoclave and store in the dark
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Now that we've learned how to assemble our sequences, we'll next learn how to identify genes (or open reading frames) on our new assemblies. We'll do that using a software program called Prodigal. After we've identified ORFs, we will compare those ORFs to other sequences using a variety of algorithms within the BLAST suite, as well as other sequence comparison tools.</p>
<p> </p>
<p><em>A note: it is <strong><u>extremely important</u></strong> that you keep careful track of where you are putting your files, or you may lose them. This is especially the case for your projects. I recommend keeping a careful list, either in a notebook or on a document on your computer, that keeps track of where all of your important files are and what directories they are in. </em></p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol outlines the analysis used to plot contig coverage statistics, as well as sequence count and length stats. We begin with visualizing contig length vs coverage. We then visualize the distributions of sequence counts per sample as a probability density plot (similar idea as a histogram), and then do the same for median sequence length. Based on the methods from the following publication:<br /><br />Hannigan, Geoffrey D., et al. "The Human Skin Double-Stranded DNA Virome: Topographical and Temporal Diversity, Genetic Enrichment, and Dynamic Associations with the Host Microbiome." <em>mBio</em> 6.5 (2015): e01578-15.<br /><br /><br />

[BEFORE_START]
Supplemental information available at:<br /><br /><a href="https://figshare.com/articles/The_Human_Skin_dsDNA_Virome_Topographical_and_Temporal_Diversity_Genetic_Enrichment_and_Dynamic_Associations_with_the_Host_Microbiome/1281248" target="_blank">https://figshare.com/articles/The_Human_Skin_dsDNA_Virome_Topographical_and_Temporal_Diversity_Genetic_Enrichment_and_Dynamic_Associations_with_the_Host_Microbiome/1281248</a>

[GUIDELINES]
<span class="identifier">sessionInfo</span><span class="paren">(</span><span class="paren">)<br /><br />## R version 3.2.0 (2015-04-16) <br />## Platform: x86_64-apple-darwin13.4.0 (64-bit) <br />## Running under: OS X 10.10.4 (Yosemite) <br />## ## locale: <br />## [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8 <br />## <br />## attached base packages: <br />## [1] stats graphics grDevices utils datasets methods base <br />## <br />## loaded via a namespace (and not attached): <br />## [1] magrittr_1.5    formatR_1.2    tools_3.2.0    htmltools_0.2.6 <br />## [5] yaml_2.1.13    stringi_0.4-1    rmarkdown_0.7    knitr_1.10.5 <br />## [9] stringr_1.0.0    digest_0.6.8    evaluate_0.7</span>

[STEPS]
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