eamag/protocols-with-steps huggingface dataset:
TITLE: RMP
AUTHORS: Akshita Varma

[STEPS]
?. [How to prepare for Risk Management (PMI-RMP) Certification?]
The candidates are required to analyse the issues properly and plan solutions accordingly to deal with the situation. The following completely depends upon the analysing quality and experience of an individual. The candidates also need to keep in mind that the solutions they find to the problems arising related to management also work for other projects without any confusion. The project managers that are newly hired have to consult the knowledge management system of the company in case of queries and ask them for inputs. The examination of the PMI RMP Certification is not so easy to qualify if the individual doesn’t carry enough knowledge about management issues and if the candidate is not prompt about risk management. The examination of PMI RMP is divided into five domains to check the capability of the individuals in each field related to risk management. The individuals have to practice very hard and gather experience about the following to pass the examination and achieve the credential of PMI RMP. There are a lot of eligibility criteria to appear in the examination.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Building predictive sensors is of paramount importance in both biology and science.  Can we make a randomly wired sensor ``good enough'' at predicting its input simply by making it larger?  We show that infinitely large, randomly wired sensors are nonspecific for their input, and therefore nonpredictive of future input, unless they are close to deterministic.  Nearly deterministic, randomly wired sensors can capture $\sim 10\%$ of the predictive information of their inputs for ``typical'' environments.</p>

[STEPS]
?.
TITLE: protocol test redesign 01
AUTHORS: Maria Gul, katarina

[DESCRIPTION]
Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Integer in sapien. Praesent id justo in neque elementum ultrices. Cras elementum. Sed ac dolor sit amet purus malesuada congue. Nunc auctor. Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur? In convallis.

[STEPS]
1. Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Integer in sapien. Praesent id justo in neque elementum ultrices. Cras elementum. Sed ac dolor sit amet purus malesuada congue. Nunc auctor. Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur? In convallis. 

Etiam quis quam. Aliquam ornare wisi eu metus. Curabitur bibendum justo non orci. Pellentesque sapien. Nullam at arcu a est sollicitudin euismod. Phasellus rhoncus. Maecenas libero. Praesent vitae arcu tempor neque lacinia pretium. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Phasellus faucibus molestie nisl. Vivamus ac leo pretium faucibus.


 

2. Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Integer in sapien. Praesent id justo in neque elementum ultrices. Cras elementum. Sed ac dolor sit amet purus malesuada congue. Nunc auctor. Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur? In convallis. Etiam quis quam. Aliquam ornare wisi eu metus. Curabitur bibendum justo non orci. Pellentesque sapien. Nullam at arcu a est sollicitudin euismod. Phasellus rhoncus. Maecenas libero. Praesent vitae arcu tempor neque lacinia pretium. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Phasellus faucibus molestie nisl. Vivamus ac leo pretium faucibus.
2.1. SUB-STEP

Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Integer in sapien. Praesent id justo in neque elementum ultrices. Cras elementum. Sed ac dolor sit amet purus malesuada congue. Nunc auctor. Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur? In convallis. Etiam quis quam. Aliquam ornare wisi eu metus. Curabitur bibendum justo non orci. Pellentesque sapien. Nullam at arcu a est sollicitudin euismod. Phasellus rhoncus. Maecenas libero. Praesent vitae arcu tempor neque lacinia pretium. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Phasellus faucibus molestie nisl. Vivamus ac leo pretium faucibus.
3. A STEP
4. THE SECOND
5. THIRD STEP
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Method used to perform quality control on Illumina sequences - includes adapter/primer removal and quality trimming<br />Goal: Remove low quality sequences to increase average quality score of reads above 28

[BEFORE_START]
**There are many tools available for this process - this is one example that has been used effectively**<br />Required software:<br />Cutadapt - https://pypi.python.org/pypi/cutadapt<br />Trimmomatic - http://www.usadellab.org/cms/?page=trimmomatic<br />Recommended software:<br />FASTQC - http://www.bioinformatics.babraham.ac.uk/projects/fastqc/

[STEPS]
?. 
?. 
?. 
?. 
?. 
?.
TITLE: CAMbank: CPT Field Processing v1
AUTHORS: Eliah G Overbey, Krista A Ryon, JangKeun Kim, Christopher E Mason

[DESCRIPTION]
Field processing of CPT vacutainers for the Cornell Aerospace Medicine Biobank (CAMbank).

Instructions for preserving: Plasma, PBMCs, and RBC Pellets.

[STEPS]
SECTION: Perform Venipuncture
1. After venipuncture, invert the tubes 8 to 10 times to fully mix in the sodium heparin anticoagulant.

Store the tube upright at room temperature until centrifugation.

To ensure proper barrier formation, blood samples should be centrifuged within 2 hours of blood collection. Centrifugation more than 2 hours after specimen collection may cause incomplete barrier formation.
SECTION: Centrifuge Settings
2. Note: A swing bucket centrifuge is required. 

Set centrifuge:
acceleration: 9
deceleration: 0
temperature: RT
duration: 30 minutes
speed: 1800xg
SECTION: Centrifuge Settings
3. Place the CPTs in the centrifuge. 
Place a protective cover over the swing buckets in case of tube breakage. 
Start the centrifuge. 
Stand by the centrifuge until the centrifuge reaches max speed. Listen for signs of imbalance or compromised tube integrity. 
SECTION: Aliquot Plasma
4. Carefully remove CPTs from the centrifuge and inspect for separation of red blood vs PMBCs vs plasma layers. 

Transfer CPTs to a sanitized laminar flow hood. 
SECTION: Wash PBMCs
6. Prepare the wash buffer (PBS + 2% FBS)

In a 50mL conical tube, mix:
24.4mL of PBS
600µL of FBS

Create more wash buffer as needed (depending on the number of tubes drawn). 
SECTION: Wash PBMCs
7. Add 5mL PBS + 2% FBS to each CPT tube.

Gently resuspend the peripheral blood mononucleocyte cells (PBMC) layer into the PBS + 2% FBS using a serological pipet.
SECTION: Aliquot Plasma
5. Using a P1000, carefully aliquot plasma (yellow layer above buffy layer) into 2D barcoded tubes at 500µL each. 

Do not pipet close enough to disturb the buffy coat (the cloudy PBMC cell layer).

Return 2D tubes with plasma to the rack and place in the -80C freezer.
SECTION: Wash PBMCs
8. One CPT tube at a time, transfer the resuspended cells into a new 15mL conical tube.
SECTION: Wash PBMCs
9. Add fresh wash buffer to bring the conical tube volume to 15mL. 
SECTION: Wash PBMCs
10. Repeat steps  8-9 for all CPTs.
SECTION: Wash PBMCs
11. Pellet the PBMCs in the centrifuge. 

Set centrifuge:
acceleration: 9
deceleration: 9
temperature: RT
duration: 15 minutes
speed: 300xg


SECTION: Wash PBMCs
12. Return the 15mL conical tubes to the laminar flow hood. 
Gently aspirate the media away from the cell pellet. 
SECTION: Viably Freeze Cells
13. Prepare freezing media: 10% DMSO, 90% FBS. 
One CPT will require 6mL of freezing media.

SECTION: Viably Freeze Cells
14. Resuspend the cells of each 15mL conical tube in 6mL of freezing media. 
SECTION: Viably Freeze Cells
15. Transfer 1mL of PBMCs into each cryovial. 
SECTION: Viably Freeze Cells
16. Place the cryovials into Mr. Frosty containers.
SECTION: Viably Freeze Cells
17. Store the Mr. Frosty's at -80C overnight, then ship back to WCM for liquid nitrogen storage. 
18. Recap and freeze the CPT tubes to preserve DNA in the red blood cell pellet
TITLE: Relative Telomere Length Measurement
AUTHORS: Sinan Gültekin

[DESCRIPTION]
Relative Telomere Length Measurement by qPCR

[STEPS]
SECTION: Relative Telomere Length Measurement
1. DNA isolation
SECTION: Relative Telomere Length Measurement
1.1. Genomic
DNA was extracted using the DNeasy Blood and Tissue Kit
(Qiagen, Hilden, Germany
SECTION: qPCR
2. 10 ng/μL DNA was amplified in a 5x EvaGreen‱ mix (No ROX) (Bio&Sell, Nurnberg, Germany). The thermal cycling protocol of  Telomere A: 5′-CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT-3′, Telomere B: 5′-GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT-3′ and B2M was: 95°C for 15 min, followed by 40 cycles of 95°C for 15 s, 56°C for 20 s, and 72°C for20 s. The thermal cycling protocol of Telomere Primer Pair 1 was:95°C for 15 min, followed by 2 cycles of 15 s at 94°C, 1 cycle of 15 s at 49°C,40 cycles of 15 s at 94°C, 1 cycle 10 s at 62°C, 1 cycle 15 s at 74°C with signal acquisition, 10 s at 84°C, and 15 s at 88°C with signal acquisition. A no-template control (NTC) and the all templates were run in triplicate for each
reaction
SECTION: qPCR
2.1. Primer sequences
Relative telomere length (RTL) was determined using two different primer pairs for telomere amplification and a primer pair for the single-copy reference gene beta-2-microglobulin(B2M). The following primers were employed: Telomere Primer Pair 1 (Hermann C, 2023): Telg: 5′-ACACTAAGGTTTGGGTTTGGGTTTGGGTTTGGGTTAGTGT-3′,Telc:
5′-TGTTAGGTATCCCTATCCCTATCCCTATCCCTATCCCTAACA-3′. Telomere Primer Pair 2 (Mugdha, 2020): Telomere A: 5′-CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT-3′, Telomere B: 5′-GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT-3′. Single Copy Gene: Beta-2-microglobulin (B2M): Forward (F): 5′-CAAGACACCCGCCAGAAGAT-3′, Reverse (R): 5′-CAGCGTGGGACAGAAGGTAG-3′ (Designed by our lab).
SECTION: Data analysis
3. Data were analyzed using ViiA7 software (ThermoFisher, MA, USA), and RTL was calculated using the ΔΔCt method: ΔΔCt = (Sample B2M Ct - Sample Telomere Ct) - (Control B2M Ct - Control Telomere Ct).
TITLE: Support Protocol 2: Metagenotyping with a Custom Collection of Genomes
AUTHORS: miriam.goldman  , chunyu.zhao  

[DESCRIPTION]
This protocol describes how to build a MIDASDB from a custom collection of genomes and perform SNV metagenotyping with it. Other MIDAS2 commands can also be run with the new database. Single-sample SNV metagenotyping is shown as an example. There are three steps: construct a custom rep-genome database for a collection of representative genomes of interest, build a bowtie2 index, and execute run_snps command with the prebuilt bowtie2 index.

[STEPS]
1. Download the example genome collection folder from Zenodo to the work directory (midas2_protocol)
 

 
MIDAS2 reserves the --midasdb_name newdb for building any new MIDASDB, and the custom MIDASDB will be built at --midasdb_dir midasdb_custom
2. Construct rep-genome component of MIDASDB
Annotate all the genomes and build the files needed for the rep-genome database. These commands should be executed in the work directory midas2_protocol.
 

 
There are two command-line parameters that users need to pass:
--debug: keep the local file after successfully build the database
--force: re-build the database even if one already exists locally
3. Build one bowtie2 index with the representative genomes.
 
We build therep-genome bowtie2 index for the two species specified via --species_list to the local directory bt2_index_custom/. Note we need to provide the custom MIDASDB midasdb_custom/ to –-midasdb_dir.
Users can also specify --bt2_indexes_name pangenomes specify to build the bowtie2 index for pangenomes.
4. Execute run_snps with the rep-genome database. For each sample, this code performs single-sample SNV calling for all the species in the bowtie2 database without any species filters (--select_threshold=-1). The number of CPUs used is specified via --num_cores 8. 
 

5. Confirm midas2 run_snps has finished successfully.
Once the single-sample SNV analysis is complete without any reported error, check for the output files (see Basic Protocol 3).
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Adipogenic differentiation was induced by using the StemPro adipogenesis differentiation kit (Invitrogen, Carlsbad, CA, USA). WJCMSCs were grown in the adipose-inducing medium for 3 weeks. For Oil Red O staining, after induction, cells were fixed with 10% formalin for at least 1 h at room temperature. Next, cells were stained with the 60% Oil Red O in isopropanol as working solution for 10 min. The proportion of Oil Red O-positive cells was determined by counting stained cells under a light microscope. The final OD value in each group was normalized with the total protein concentrations prepared from a duplicate plate.</p>

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

[GUIDELINES]
<strong>NOTE: We observe maximal growth with open ocean seawater (i.e. Sargasso seawater), though we can routinely grow <em>Prochlorococcus</em> in some coastal seawater (from Cape Cod, MA) as well.<br /><br />Table 1.</strong> Nutrient additions to filtered, autoclaved seawater <br />
<table style="height: 384px;" width="751">
<tbody>
<tr>
<td><strong>Nutrient</strong></td>
<td><strong>Manufacturer/Grade&nbsp;</strong></td>
<td><strong> Primary Stock (M)&nbsp;</strong></td>
<td><strong> Dilution Factor&nbsp;</strong></td>
<td><strong> Final Conc. (&micro;M) </strong></td>
<td><strong>Sigma Cat. No.</strong></td>
</tr>
<tr>
<td>NaH<sub>2</sub>PO<sub>4</sub>&middot;H<sub>2</sub>O&nbsp;</td>
<td>(1) / ACS&nbsp;</td>
<td>0.025&nbsp;</td>
<td>1:500&nbsp;</td>
<td>50</td>
<td>(MK7892-04)</td>
</tr>
<tr>
<td>NH<sub>4</sub>Cl&nbsp;</td>
<td>(1) / ACS</td>
<td>0.50&nbsp;</td>
<td>1:625&nbsp;</td>
<td>800</td>
<td>(MK3384-12)</td>
</tr>
<tr>
<td>Na<sub>2</sub>EDTA&middot;2 H<sub>2</sub>O&nbsp;</td>
<td>(2) / 99%&nbsp;</td>
<td>0.012&nbsp;</td>
<td>1:10<sup>4</sup>&nbsp;</td>
<td>1.17</td>
<td>E4884-100g</td>
</tr>
<tr>
<td>FeCl<sub>3</sub>&middot;6 H<sub>2</sub>O&nbsp;</td>
<td>(1) / Analytic&nbsp;</td>
<td>0.012&nbsp;</td>
<td>1:10<sup>4</sup>&nbsp;</td>
<td>1.18</td>
<td>44944-50g</td>
</tr>
<tr>
<td>ZnSO<sub>4</sub>&middot;7 H<sub>2</sub>O&nbsp;</td>
<td>(3) / &gt;99.5%&nbsp;</td>
<td>0.080&nbsp;</td>
<td>1:10<sup>7</sup>&nbsp;</td>
<td>0.008</td>
<td>204986-10g</td>
</tr>
<tr>
<td>CoCl<sub>2</sub>&middot;6 H<sub>2</sub>O&nbsp;</td>
<td>(1) / Analytic&nbsp;</td>
<td>0.050&nbsp;</td>
<td>1:10<sup>7</sup>&nbsp;</td>
<td>0.005</td>
<td>60820-50g</td>
</tr>
<tr>
<td>MnCl<sub>2</sub>&middot;4 H<sub>2</sub>O&nbsp;</td>
<td>(1) / Analytic&nbsp;</td>
<td>0.900&nbsp;</td>
<td>1:10<sup>7</sup>&nbsp;</td>
<td>0.090</td>
<td>M3634-100g</td>
</tr>
<tr>
<td>Na<sub>2</sub>MoO<sub>4</sub>&middot;2 H<sub>2</sub>O</td>
<td>(2) / ACS&nbsp;</td>
<td>0.030&nbsp;</td>
<td>1:10<sup>7</sup>&nbsp;</td>
<td>0.003</td>
<td>M1651-100g</td>
</tr>
<tr>
<td>Na<sub>2</sub>SeO<sub>3</sub>&nbsp;</td>
<td>(2) / ~98%&nbsp;</td>
<td>0.100&nbsp;</td>
<td>1:10<sup>7</sup>&nbsp;</td>
<td>0.010</td>
<td>S5261-25g</td>
</tr>
<tr>
<td>NiCl<sub>2</sub>&middot;6 H<sub>2</sub>O&nbsp;</td>
<td>(1) / Analytic&nbsp;</td>
<td>0.100&nbsp;</td>
<td>1:10<sup>7</sup></td>
<td>&nbsp;0.010</td>
<td>223387-25g</td>
</tr>
</tbody>
</table>
Manufacturer Index: (1) Mallinckrodt, (2) Sigma, (3) Fluka<strong> Note 2/6/12:</strong> Mallinckrodt and Fluka no longer available; ordered all reagents from Sigma at same grade as specified.<br /><br /><strong>Note 4/28/08:</strong> <br />Matt suggested higher concentrations of NH<sub>4</sub>Cl and NaH<sub>2</sub>PO<sub>4</sub> so only use 0.5ml per 1L seawater:<br />1.6M NH<sub>4</sub>Cl &nbsp; &nbsp; 2.14g/25ml or 8.56g/100ml<br />0.1M NaH<sub>2</sub>PO<sub>4</sub>-H<sub>2</sub>O &nbsp; &nbsp; 0.345g/25ml or 1.38g/100ml<br />Trace metal working stock 100&micro;l

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Collecting the Red Turpentine Beetle
AUTHORS: Caroline Storer, Jiri Hulcr

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to collect and preserve the red turpentine beetle (RTB), Dendroctonus valens.</div><div class = "text-block"><span>This protocol is part of the Bark Beetle Mycobiome (BBM) Research Coordination Network. For more information on the BBM international network: Hulcr J, Barnes I, De Beer ZW, Duong TA, Gazis R, Johnson AJ, Jusino MA, Kasson MT, Li Y, Lynch S, Mayers C, Musvuugwa T, Roets F, Seltmann KC, Six D, Vanderpool D, & Villari C. 2020. Bark beetle mycobiome: collaboratively defined research priorities on a widespread insect-fungus symbiosis. Symbiosis 81: 101–113 </span><a href="https://doi.org/10.1007/s13199-020-00686-9" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1007/s13199-020-00686-9</span></a><span>.</span></div></div>

[STEPS]
TITLE: Sanger Tree of Life Sample Homogenisation: PowerMash
AUTHORS: Amy Denton, graeme oatley, Clare Cornwell, Michael Quail, Caroline Howard

[DESCRIPTION]
This protocol describes the homogenisation of tissue samples for DNA and/or RNA extraction intended for long read sequencing or RNA-Seq, using the Diagnocine PowerMasher II tissue disruptor. This process is highly effective for the disruption of tissue samples weighing less than 25 mg from all taxonomic groups covered by the Tree of Life Programme except for plants, fungi, protists, sponges and corals. Larger samples are not processed through this method due to difficulties in achieving homogenisation. Crustaceans and molluscs are particularly challenging samples. The output of this protocol is a sample that can be directed toward any of the Sanger Tree of Life DNA and RNA extraction protocols.

[BEFORE_START]
Prepare lysis buffer for the tissue’s intended downstream protocol: either DNA or RNA extraction.

[GUIDELINES]
Keep tissues on dry ice prior to transfer into the BioMasher tube and lysis buffer addition in order to maintain a low temperature and prevent nucleic acid degradation.
This method is not recommended for samples that weigh more than 25 mg.
This method is not recommended for plants, fungi, moss, protists, sponges or corals samples –  these samples should be disrupted using a different method.

[STEPS]
SECTION: Laboratory protocol
1. Transfer the sample into the sterile 1.5 mL BioMasher II tube and add the lysis buffer required for the downstream protocol.
SECTION: Laboratory protocol
2. Attach the sterile BioMasher pestle to the PowerMasher II tissue disruptor, avoiding contact with the end of the pestle that will contact the sample to prevent contamination.
SECTION: Laboratory protocol
3. Disrupt the sample in the lysis buffer within the BioMasher tube using the PowerMasher II tissue disruptor and sterile BioMasher pestle; this is done by placing the pestle into the BioMasher tube and against the sample, applying some gentle pressure so the sample remains between the pestle and the wall of the BioMasher tube, and then squeezing the trigger of the PowerMasher II tissue disruptor to activate it.
SECTION: Laboratory protocol
4. Continue for approximately 30 seconds to 1 minute (depending on the tissue type), until no large pieces of the sample remain, or the sample cannot be disrupted any further. Gently moving the pestle up and down can encourage sample movement within the lysis buffer and prevents the sample becoming stuck at the bottom of the tube, allowing for maximum disruption.
SECTION: Laboratory protocol
5. Remove the pestle from the BioMasher tube and disconnect it from the PowerMasher tissue disruptor II. If any tissue remains stuck to the pestle, gently try to remove this by wiping the pestle tip on the rim of the BioMasher tube. Dispose of pestle in biological waste.
SECTION: Laboratory protocol
6. Proceed with the powermashed sample to the next step of the tissue’s downstream protocol  – 
 either DNA or RNA extraction.
TITLE: Preparation of cigarette smoke extract (CSE)
AUTHORS: Yoshiyuki Matsuo, Kiichi Hirota

[STEPS]
?. [Notes]
All procedures should be done in a fume hood to minimize exposure of the experimenter and laboratory environment to the cigarette smoke.The CSE should be prepared immediately before use.
?. [Materials]
1000 µl pipette tipSilicone tubing (Tigers Polymer, SR-1554, inner diameter: 3 mm, outer diameter: 5 mm, length: 250 mm)Polypropylene tubing (inner diameter: 2 mm, outer diameter: 4 mm, length: 150 mm)Polypropylene tubing (inner diameter: 5 mm, outer diameter: 7 mm, length: 75 mm)Silicone stopper with two holes50 ml polypropylene conical tube (Thermo Fisher Scientific, 339657) Extension tubing (Terumo, SF-ET5527, 750 mm)Three-way stopcock (Terumo, TS-TR2K)50 ml syringe (Terumo, SS-50LZ)phosphate-buffered saline (PBS)Millex-LG sterilizing filter unit (Merck/Millipore, SLLG025SS, 0.2 µm)Cigarettes
?. [Protocol]
Assemble the smoking apparatus.
?. [Protocol]
Fill the 50 ml polypropylene conical tube with 10 ml of PBS (6), and cap it with the silicone stopper (5).
?. [Protocol]
Attach the 50 ml syringe (9) to the 3-way stopcock (8) connected to the extension tubing (7).
?. [Protocol]
Insert the cigarette into the 1000 µl pipette tip (1), and attach it to the silicone tubing (2).
?. [Protocol]
Light the cigarette while pulling on the syringe plunger.
?. [Protocol]
Draw the syringe plunger to the 50 ml mark in 10 seconds (flow rate: 300 ml/min). The mainstream smoke is bubbled through PBS in the conical tube.
?. [Protocol]
Turn the stopcock off to the extension tubing and push the plunger back into the syringe.
?. [Protocol]
Open the stopcock to the extension tubing.
?. [Protocol]
Repeat the steps 8-10 until the cigarette burns to the filter (typically 10 to 11 times per cigarette).
?. [Protocol]
Remove the cigarette butt and insert the new cigarette to the 1000 µl pipette tip for the next round of extraction.
?. [Protocol]
Repeat the procedure for a total of 5 cigarettes (smoke from 5 cigarettes is bubbled through 10 ml of PBS).
?. [Protocol]
Filter the extract through the 0.2 µm pore size membrane, and the resulting solution is defined as 100% CSE.
TITLE: Short term effect of Aldicarb on C.elegans
AUTHORS: Priota Islam

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Aldicarb is a cholinesterase inhibitor which prevents the breakdown of acetylcholine in the synapse​</li><li style = "counter-reset:ol0;list-style-type:disc;">Reported to paralyze C. elegans​</li><li style = "counter-reset:ol0;list-style-type:disc;">Strains of interest believed to be resistant to the paralytic effect​</li><li style = "counter-reset:ol0;list-style-type:disc;">Strains: OW1601 & OW1603​
​</li></ul></div></div>

[STEPS]
?. Bleach synchronize the worms on a Friday
?. Following Monday refeed the arrested L1s on OP50 seeded plates, make 2plates per strain keeping one plate at 20C and the other at 25C
?. The day before tracking (Wednesday) add 35ul of 2.5mM AK to 24 imaging plates
?. Also, seed those drugs treated 24 imaging plates and additional 24 non-treated imaging plates with 50ul of 1:10 OP50 and leave to dry O/N
?. On the day of tracking (Thursday), transfer 5 worms per strain on to the aldicarb treated plates and leave for 1.5hrs, pick onto the controlled plates too
?. After 1.5hrs image for 15mins on PhenixTrack 8 sets: (2 treatment sets + 2 control sets with no treatment) x 2 growth temperatures