eamag/protocols-with-steps huggingface dataset:
TITLE: New Iron Extracting Method from Cattle's Blood for Iron Concentration Analysis
AUTHORS: Khairil Asnan, Mohd. Sadzan Asram bin Aliming, Aprialdy Idrus, Diana Eka Pratiwi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify"><span>Cattle's blood component is compressed using centrifuge (5000 rpm for 10 minutes), then supernatant (erythrocytes) collected. Erythrocytes with a ratio of 3:1 (serum:supernatant), treated with initial mixing with NaOH (0.5 M) with an initial ratio 1:1 (v/v) and let it sit for 30 seconds then mixed it by centrifugation about 10 rpm for 30 seconds. Sample then treated by mixing oleic acid (2:1 v/v). The sample then dehydrated by heat about 121</span><span style = "vertical-align:super;">o</span><span>C for a week. Calculated total iron mass was 240000 μg/100 ml whole blood sample (about 14.40% content of the whole sample). Sample puriﬁed by furnace using high temperature about 800</span><span style = "vertical-align:super;">o</span><span>C for 2 hours and increased the iron concentration up to 46.30% (m/m%).</span></div><style>
							  .justify:after {
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[STEPS]
?. [Sample preparation]
Compress the whole blood sample from cattle.
6 ml
37 °C
?. [Sample preparation]
Mix  sample with
[blood sample]
[NaOH]
37 °C
dark green solution with strong odor
?. Chelate reaction by adding  into the previous mixed sample solution then let it sit for . After that, mixed them by  for  .
[Oleic acid]
TITLE: RNAi by feeding in Euplotes focardii (povisional)
AUTHORS: Francesca Papi, Angela Piersanti

[STEPS]
?. Grow RNAse III deficient E.coli strain HT115 in LB with antibiotic selection o/n at 37°C.
?. Prepare a 1:100 dilution of the bacterial culture, and grow it at 37°C until it reaches an OD600 of 0.4.
?. Add 0.4 mM IPTG, and induce RNA transcription from the L4440 plasmid, in which it is included the gene of interest  to be silenced, in the bacteria at 37°C, until they reach a OD600 of 1. Bacteria with  L4440 plasmid, empty vector (EV), were grown as well to be used as control.
?. Collect the bacteria by centrifugation at 7000 rpm for 8 minutes, wash them twice with ddH2O, then resuspend in 1.5 ml of  sea water (at 4°C) to an OD600 of 4 to feed  100 ml of Euplotes focardii culture with a cell density of ∼3000 cells/ml.
?. Feed Euplotes focardii culture every 3 days, at least for 10 days and checked the phenotype.
TITLE: VISUALIZING PROTEINS ON PAGE GEL WITH COOMASSIE COLLOIDAL STAIN - CHEM 584
AUTHORS: Ken Christensen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Staining polyacrylamide gels using a colloidal Coomassie Blue stain. Adapted from a JoVE article.</div></div>

[STEPS]
?. Wash gel with ~50 mL ddH2O 3x for ~ for each wash on a shaker NOTE: insufficient washing causes poor sensitivity because the remaining SDS on the gels disturbs the bounding of the dye to the protein
?. Shake the colloidal Coomassie solution before use to disperse particles evenly.
?. Incubate the gels with the Coomassie solution by agitation on a shaker up to . This staining generates marginal background so you can observe the staining progress.Note: after 10 minutes you should see some protein bands appearing, within 2 hours of incubation about 80% maximum staining is completed. For nearly maximum sensitivity, an overnight incubation is recommended. In some cases, when the amount of protein to be stained is large, the solution turns a bright blue color. If this happens, you should refresh the staining solution as necessary.
?. After staining, remove the Coomassie solution and rinse the gels twice with ddH2O.
Note: you can reuse the staining solution as long as particles still remain, otherwise discard it appropriately.
?. Remove any dye particles from the staining dish with a lint-free paper towel and destain for up to  in ddH2O or a Coomassie destain solution (10% (v/v) 96% ethanol, 2% (v/v) orthophosphoric acid 85% in ddH2O.)
?. Finally rinse the gels twice with ddH2O: the gels will reach their original thickness (the alcohol-acid media makes the gels shrink). Neutralization in water also enhances the Coomassie stain’s color intensity.
?. You can record your gel using the gel documentation system or scan it on the Licor using the 680/700 nm channel.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol is part of the VERVE holiday drive to collect exotic off-the-shelf laboratory recipes. More details <a href="https://www.protocols.io/g/verve-net/news/call-for-recipes" target="_blank">here</a>.

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

[DESCRIPTION]
<p>Instructions to make 50 mL of incubation buffer for protoplast isolation.</p>
<p>Adapted from Yoo et al. 2007 http://www.nature.com/nprot/journal/v2/n7/full/nprot.2007.199.html</p>

[STEPS]
?. 
?. 
?.
TITLE: Patient PBMC collection and cryopreservation and cryorecovery
AUTHORS: Rebecca Wallings

[DESCRIPTION]
Isolation of PBMC from human blood using CPT tubes, cryopreservation and cryorecovery of cells

[STEPS]
SECTION: PBMC collection and cryopreservation
1. Collect 8 mL of blood per BD Vacutainer CPT Cell Preparation Tube with Sodium Citrate (BD Biosciences, 362761). 

Invert tubes 8-10 times and centrifuged at room temperature at 1500x g for 20 minutes with no brake. 

Using a 10mL stripette , transfer the PBMC enriched layer to a new 50-mL conical tube, being sure not to touch the ficoll layer, and add MACS buffer (PBS, 0.5% bovine serum albumin, 20 mM EDTA, pH 7.2) to a final volume of 50 mL, followed by centrifugation at 1800x g for 10 minutes at room temperature.

Following removal of the supernatant, resuspend PBMCs in 10mL MACS buffer and count using a hemocytometer with Trypan blue at a 1:20 dilution (10uL of cells mixed with 10uL of 1:20 Trypan blue solution). 

Centrifuge PBMCs for 10 min at 300x g at 4°C.

Aspirate supernatant and gently resuspend cell pellet in cryopreservation media (RPMI 1640, with 20% FBS and 10% DMSO) at a final concentration of 1 x 77 cells/mL in cryovials (Simport, T311-2).

Place cryovials in a room-temperature Mr. Frosty freezing container with isopropanol as per manufacturer’s instructions and store at -80°C overnight. 

Remove cryovials from freezing containers and immediately placed into liquid nitrogen for long-term storage. 
SECTION: Cryorecovery
2. For cryorecovery, retrieve PBMCs from liquid nitrogen, and thaw at 37°C in water bath. 

Once thawed (roughly 1 minute with agitation) slowly add contents of one cryovial to 25mL 37°C filter sterilized complete culture media (RPMI 1640 media, 10% heat-inactivated FBS, 1mM Penicillin-Streptomycin) in 50mL falcon and pellet via centrifugation at 300x g for 10 min at room temperature. 

Remove supernatant and resuspend cells in 37°C complete culture media for cell counting using Trypan blue.
TITLE: Magnitude of job satisfaction and intention to leave present job among nurses in selected Federal Hospitals in Addis Ababa, Ethiopiantitled collection
AUTHORS: Aynye   Negese*,   Elsabet Getye2, ziyadahm  

[DESCRIPTION]
Background 

Job dissatisfaction issues and Health workers’ intention to leave is an increasing problem that threatens the function of the health care sector worldwide, especially in developing countries, including Ethiopia. As part of future evidence, this study was intended to assess nurses’ job satisfaction and intention to leave their current work and its associated factors in federal public hospitals in Addis Ababa, Ethiopia. 
Method

An institutional-based cross-sectional study design was used to conduct the study. A simple random sampling technique was employed to select 408 nurses from selected federal Hospitals in Ethiopia, and used self-administered questioner to collect the data. Logistic regression was employed to identify the predictor variables with consideration of a statistical significance of P <0.05 adjusted odds ratios calculated at 95% CIs.
Result

The magnitudes of job satisfaction and intention to leave current job among nurses in this study were 47.7% and 80.6%, respectively. Salary is imbalance with demands [AOR=2.85 (1.24, 6.57)], managers who have no a personal plan for developing skills [AOR=3.74 (1.58, 8.87)], stressful jobs [AOR=0.28 (0.11, 0.71)],health problem is a reason for having thoughts about changing jobs [AOR=3.02 (1.17, 7.79)] and a lack of development [AOR=4.13 (1.51, 11.3)]were identified determinant factors for intention to leave.
Conclusion

The overall intention to leave current job among nurses working in selected federal Hospitals in Ethiopia was high. The government of Ethiopia should balance salary of the nurses with the current market level. The hospitals leaders should plan the way nurses develop their educational and job carrier level. 
Key words: Job satisfaction, Intention to leave, turnover, nurses, hospitals, Addis Ababa

[STEPS]
TITLE: cDNA Library Preparation for scRNA-seq (10x Genomics) 
AUTHORS: molmer  , Martin  Lotz, Tony  Mondala, Steven  Head

[DESCRIPTION]
Samples are processed using V2 barcoding chemistry kits of 10x Genomics. For each run, 10,000 cells from individual donors are labeled with distinct oligo-barcoded antibodies (cell hashing), enabling us to pool samples from both conditions on each 10X Genomics run and reduce batch effects. All samples from a given experiment are processed in parallel in the same thermal cycler. The 10x user guide can be found https://assets.ctfassets.net/an68im79xiti/1C16trEdzy1Folq5xbOijE/7e6fb1f504e130bd561d898384da99d9/CG000315_ChromiumNextGEMSingleCell3-_GeneExpression_v3.1_DualIndex__RevB.pdf and is also an attached document.

[STEPS]
1. Single cells are isolated from human femoral articular cartilage using doi 
dx.doi.org/10.17504/protocols.io.14egn765qv5d/v1.
2. Single cell suspensions are multiplexed and converted to barcoded scRNAseq libraries using the Chromium Single Cell 3’ Library, Gel Bead and Chip Kit (10x Genomics), and the BD™ Hu Single Cell Sample Multiplexing Kit.
 


3. Libraries are sequenced on an Illumina NextSeq2000 sequencer targeting 20,000-25,000 reads per cell.
TITLE: Oprah Winfrey ACV Gummies Reviews: Shark tank gummy candy 2022
AUTHORS: oprahwinfreyreviewsads  

[DESCRIPTION]
Oprah Winfrey ACV Gummies Reviews: Shark tank gummy candy 2022


[STEPS]
1. Oprah Winfrey ACV Gummies Reviews: Shark tank gummy candy 2022

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https://www.inkitt.com/stories/romance/911012/chapters/2
TITLE: Luminex Bead Conjugation
AUTHORS: Andrew Crowley

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for amine-coupling of proteins to magnetic Luminex beads</div><div class = "text-block"><span>Quantity as written: 1 x 10</span><span style = "vertical-align:super;">6</span><span> beads under typical yields (sufficient for 2000 wells assuming 500 beads/well)</span></div><div class = "text-block">### Needs a good qualification section!</div></div>

[STEPS]
?. [Activation]
Resuspend beads by briefly vortexing; dispense
[(contains approx. 1.25 million beads)]
?. [Activation]
BEAD SEPARATION BLOCK
?. [Activation]
Collect volume by pulse centrifugation
?. [Activation]
Place on magnetic tube rack, shielded from light
Protocol tipPartially open the microcentrifuge tube before placing in the rack. This allows them to be opened on-rack with minimal disruption of the bead pellet that's held in place only by the magnet.
?. [Activation]
Remove   of supernatant (while still on-rack) using a pipette
90 µl
n.b.This leaves a   excess that allows the removal of supernatant at all subsequent steps to use the same volume that was added immediately prior.
?. [Activation]
WASH BLOCK
?. [Activation]
Resuspend in  1x PBS; vortex briefly to mix
100 µl
?. [Activation]
Collect volume by pulse centrifugation
?. [Activation]
Place on magnetic tube rack, shielded from light
?. [Activation]
Remove supernatant using a pipette
?. [Activation]
Resuspend beads in a solution containing:    -  sodium phosphate (pH 6.2)     -  (approx.  )  in sodium phosphate (pH 6.2)     -  (approx.  ) sulfo-NHS in phosphate (pH 6.2)
80 µl
5 µl
5 µl
?. [Activation]
Mix tubes end-over-end, shielded from light
0 Room temperature
?. [Coupling]
(separation block) for removal of supernatant
?. [Coupling]
Resuspend beads in  protein of interest , made in 1x PBS
250 µl
?. [Coupling]
Mix tubes end-over-end, shielded from light
0 Room temperature
?. [Blocking + Washes]
(wash block) for a total of 3 washes using  1x PBS-TBN
400 µl
n.b.DO NOT use P1000 for removing supernatant as the wider tip and faster flow tends to lead to greater bead loss. Instead, use a P200 twice.
?. [Blocking + Washes]
Perform final resuspension in  1x PBS-TBN
250 µl
?. [Blocking + Washes]
Will the beads be used immediately?
?. [Counting]
Dilute beads by 1:10 and 1:100 in 1x PBS
?. [Counting]
Measure concentration of each dilution by cell counter
Typical yields are appox. 1 x 106 beads, or 4 x 106 beads/mL
TITLE: 08 Exploration of expression condition
AUTHORS: TJUSLS China

[STEPS]
?. Transform the plasmid into bacteria used to express target protein(e.g. E.coli BL21(DE3)).
?. Take monoclone in the culture plate into LB tube and cultivate in shaking incubator overnight(10-12h) to activate bacteria.
?. Test the OD600 number of bacteria, then pipe 5-10ul into each new 5 mL LB tube. Don’t forget to add antibiotic into tubes and mark them.
?. Cultivate in shaking incubator for 3-4 hours until the OD600 of bacteria range from 0.6 to 0.8.
?. Pipet 200ul bacterial liquid as uninduced sample, and take another 600ul to mix with 400ul 50%glycerol to store. Then add inducer IPTG into each tube in different concentration, and incubate at 16°C for 16 hours or at 37°C for 4 hours shaking at 200-300rpm.
600 µl
?. After cultivating, pipet 200ul for each as induced sample.(The method to make samples  1) Centrifuge the taken bacterial liquid at 12,000rpm for 3 minutes  2) Drop the supernatant and resuspend the precipitate using 100ul ddw.  3) Pipet 50ul resuspending liquid to mix with 10ul 6XSDS Loading buffer  4) Boil it in dry bath at 100°C for 10 minutes)Use SDS-PAGE to check whether the target protein express or not and what the most suitable condition for its expression is.
200 µl
TITLE: How to prepare zebrafish brain tissue samples for biochemical assays
AUTHORS: Adrieli Sachett, Matheus Gallas-Lopes, Radharani , Greicy M M Conterato, Ana Herrmann, Angelo Piato

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Zebrafish are increasingly used as a model animal in neuroscience research. Here we describe our protocol to collect and process zebrafish brains so they are well presearved and viable for biochemical assays.</div></div>

[STEPS]
?. [Preparations to collect animal tissue]
Before starting to collect animal tissue, it is important to prepare some settings in order to guarantee the appropriate preservation of the sample and, ultimately, the assessment of biochemical parameters;
?. [Sample collection and processing]
The following steps should be carried out following animal welfare and ethical guidelines;
?. [Preparations to collect animal tissue]
Fill a thermal box with shaved ice;
?. [Preparations to collect animal tissue]
Prepare the  microtubes that will be used to store tissue samples with the correct information. Microtubes used in this step should have a conical bottom to ensure the homogenization of the tissue using a pestle with a conical end;
1.5 mL
?. [Preparations to collect animal tissue]
After the microtubes are correctly identified, they must be buried halfway in the ice and filled with  of phosphate buffered saline solution  at ;
150
4 °C
?. [Sample collection and processing]
Euthanize the animals by exposure to chilled water between  and  until loss of orientation and cessation of opercular movements;
2 °C
4 °C
?. [Sample collection and processing]
Two minutes after the loss of orientation and cessation of opercular movements, use a scalpel to remove the cranium of the fish to collect brain tissue;
?. [Sample collection and processing]
Place each brain in the respective microtube with the help of the scalpel, making sure the tissue is immersed in the solution and the microtube stays in the ice;
?. [Sample collection and processing]
After finishing the sample collection, use a pestle with a conical end to homogenize the tissue in the solution. Move the pestle up and down making circular movements to grind the tissue between the microtube and the pestle. Homogenize the samples for  (performing the circular movements around 60 times). The same researcher should homogenize all of the samples for better standadization of the process;
?. [Sample collection and processing]
If you are using a pool of two or more brains, add  of chilled phosphate-buffered saline solution to the tube for each additional brain;
150
?. [Sample collection and processing]
Use a vortexer to mix the samples for ;
?. [Sample collection and processing]
Centrifuge the samples ;
Centrifuge: 3000 34, 10 min, 4 10
?. [Sample collection and processing]
Use a micropipette to collect the supernatant and transfer it to a new microtube properly identified. Be careful to avoid the precipitate;
?. [Sample collection and processing]
Store your samples in a freezer at .
-80 °C
TITLE: Agar Plate Preparation
AUTHORS: Andrew Crowley

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Default scale - 1 L (makes approx. 2.5 sleeves of plates)</div></div>

[STEPS]
?. Add powdered LB ( ) and agar () to deionized water ( )
25 g
15 g
1 L
n.b.Particulates may remain after addition, but these will dissolve during the autoclave cycle
?. Autoclave using liquid cycle
?. Allow media to cool to
50 °C
?. Select antibiotic from the list below:
?. Add  kanamycin and mix thoroughly
50 mg
?. Add  ampicillin and mix thoroughly
100 mg
?. Autoclave using liquid cycle
?. Pour into sterile plates and allow to cool
?. Pour into sterile plates and allow to cool
TITLE: RNA extraction from hairy roots of common bean (Phaseolus vulgaris L.) and cDNA synthesis
AUTHORS: Ronal Pacheco Sánchez, Noreide Nava

[DESCRIPTION]
Extracting RNA for subsequent quantification of transcript levels by RT-qPCR requires high purity and concentration. When the amount of tissue is not abundant, as is the case with hairy roots, the concentration of RNA is frequently low. Here we present an optimized protocol for TRIzol-mediated RNA extraction from hairy roots of common bean. This protocol is based on the manufacturer's instructions   


[STEPS]
SECTION: Extraction of total RNA
1. Macerate root tissue using liquid nitrogen.
SECTION: Extraction of total RNA
2. Load 100 mg of macerated tissue into a 1.5 mL Eppendorf tube and add 1 mL of   . 
 





SECTION: Extraction of total RNA
3. Mix by vortexing15 s  and incubate for 5 min at room temperature. 

5. Centrifuge  11800 rpm, 15 min, 4 °C 

SECTION: Extraction of total RNA
4. Add200 µL, mix by vortexing 15 s and incubate for 3 min at room temperature.

6. Transfer the aqueous phase to a new 1.5 mL Eppendorf tube.
7. Add 500 µL of isopropanol, mix by immersion,  and incubate 10 min -20 °C .
 



8. Centrifuge 11800 rpm, 10 min, 4 °C 

9. Transfer the aqueous phase to a new 1.5 mL Eppendorf tube.
10. Add 500 µL of 4 M  LiCl and rise the pellet, do not resuspend, vortex slowly.
 

11. Centrifuge  5900 rpm, 4 °C20 min 

12. Discard the LiCl phase. 
 

13. Add  500 µL of tris-EDTA buffer pH 8 . Resuspend RNA by vortexing.

14. Add 500 µL and mix by vortexing.

15. Centrifuge 5900 rpm, 10 min, 4 °C 

16. Transfer the aqueous phase to a new 1.5 mL Eppendorf tube.
17. Add 500 µL of  isopropanol and 66 µL of 3 M sodium acetate pH 5.2  Mix by immersion and incubate   -20 °C 
 




18. Centrifuge 11800 rpm, 10 min, 4 °C 

19. Discard the aqueous phase and vacuum or air dry the RNA pellet.
20. Resuspend RNA pellets using nuclease-free water. Preferably, use DEPC-treated water. 
SECTION: Preparation of RNA samples for cDNA synthesis
21. Check the integrity of RNA in a 1% agarose gel treated with bleach. 

 .

SECTION: Preparation of RNA samples for cDNA synthesis
22. Prepare a dilution (1/10) of each RNA sample and quantify the concentration using a  or an equivalent instrument.

SECTION: Preparation of RNA samples for cDNA synthesis
23. Prepare one aliquot 10 µLof each RNA sample at 10 ng/μl

SECTION: Preparation of RNA samples for cDNA synthesis
24. Add 1 µL of   
and 1 µL of the corresponding buffer (10X) to each RNA sample.
 


SECTION: Preparation of RNA samples for cDNA synthesis
25. Incubate samples30 min  37 °C 
 



SECTION: Preparation of RNA samples for cDNA synthesis
26. Add 1 µL of 10 millimolar (mM) to each sample and incubate5 min 70 °C, then immediately incubate on ice .

SECTION: Preparation of RNA samples for cDNA synthesis
27. 
SECTION: Synthesis of cDNA
28. Prepare a mix containing 1 µL of , 4 µL of the corresponding buffer (5X), and 2 µL . Add 7 µL of the mix to each RNA sample.

SECTION: Synthesis of cDNA
29. Incubate 90 min at42 °C and 10 min 70 °C .

SECTION: Synthesis of cDNA
30. Store cDNA samples at -20 °C .