eamag/protocols-with-steps huggingface dataset:
TITLE: TS Procure 812 - primary fixation Karnovsky&#39;s - transwells (TM - 013)
AUTHORS: sandra.crameri

[DESCRIPTION]
The method is used to Karnovsky's EM fixed transwells to Procure 812 resin blocks. The presence of 4% paraformaldehyde is only used to satisfy microsecurity requirements.

[GUIDELINES]
Sample is not optimal for EM, best to dissect and use only the outer 2mm of tissue mass. Sample is usually derived from histology.

[STEPS]
SECTION: HEADER
1. SAN:




SPEC No:




OPERATOR & STEPS:




OPERATOR & STEPS:
SECTION: Conventional
2. Karnovsky's fixative: 2.5 % volume   and 4 % volume   in 0.1 Molarity (M) Sorenson's Phosphate buffer (pH 7.2, 300mosmol/kg) for at least 40 min  or overnight

SECTION: Conventional
3. 0.1 Molarity (M) buffer 15 min 

SECTION: Conventional
4. 1 % volume   in buffer 60 min 

SECTION: Conventional
5. 70 % volume   for at least 20 min  or overnight

SECTION: Conventional
6. 95 % volume    20 min 

SECTION: Conventional
7. 100 % volume    20 min 

SECTION: Conventional
8. 100 % volume    dried with molecular sieve 20 min 


SECTION: Conventional
10. 50 % volume   50 % volume   20 min 

SECTION: Conventional
11. 100 % volume   20 min 

SECTION: Conventional
12. 50 % volume   50 % volume Procure 812


SECTION: Conventional
13. 100 % volume Procure 81220 min 

SECTION: Conventional
14. 100 % volume Procure 812 60 min 

SECTION: Conventional
15. Embed & polymerise overnight in oven at 60 °C , section with diamond knife as usual.

SECTION: Remove membrane
9. Remove membrane from transwell scaffold using a 45o scalpel and transfer to a glass vial with 100 % volume 
  dried with molecular sieve.
TITLE: HEPES-Sucrose Cutting Solution
AUTHORS: Allen Institute

[DESCRIPTION]
This protocol is used for preparing HEPES-Sucrose Cutting Solution, used for animal perfusion following anesthesia.

Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

[STEPS]
TITLE: Terrific Broth Medium
AUTHORS: is Sparrow, Steven J Burgess

[DESCRIPTION]
Tartoff, K.D. and Hobbs, C.A. (1987) Improved Media for Growing Plasmid and Cosmid Clones. Bethesda Research Laboratories Focus, 9, 12.

[GUIDELINES]
Prepare the stock solutions and autoclave separately before combining when cooled to create TB medium. The reason for this is that Glycerol is believed to react with amines in the Tryptone/Yeast extract via the Malliard reaction, leading to the formation of compounds which 

[STEPS]
SECTION: Prepare stock solutions
1. Combine the following components for 1L TB buffer
24 g  
20 g    
4 mL Glycerol
Make up volume to 900 mL with dH2O

Autoclave 121 °C 30 min 

SECTION: Prepare medium
3. Allow solutions to cool to 60 °C then add
100 mL of 10x TB salts to 900 mL TB buffer.

SECTION: Prepare medium
4. Store at Room temperature 

SECTION: Prepare stock solutions
2. Prepare 1L of 10x TB Salts (0.17 M KH2PO4, 0.72 M K2HPO4) by combining the following 
23.1 g  KH2PO4
125.4 g K2HPO4
Make up volume to 1 L with dH2O

Autoclave  121 °C 30 min 


SECTION: Practical use
5. We have found that using TB for plasmid preps consistently yields higher than using LB regardless of prep kit used, with half the volume of culture required.

It is not recommended to exceed ~2 mL of saturated TB culture per prep column due to overloading the column and not yielding clean preps.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
For use in "Isolation of cyanophages by plaque assyays"

[BEFORE_START]
Prepare 100 mL portions of 0.4 to 0.5% (w/v) of purified agar, agarose or low-melting point (LMP) agarose (i.e., Invitrogen #15517-022) in your media of choice. Although LMP agarose can be quite expensive, it is recommended for temperature sensitive samples and cells, since it solidifies at ca. 25°C. Purified agars, as well as low-melting point agars and agaroses are available for a range of lower temperatures (consult the following websites for more details:<a href="http:// www.sigmaaldrich.com" target="_blank"> www.sigmaaldrich.com</a> and <a href="http://www.invitrogen.com" target="_blank">www.invitrogen.com</a>)

[STEPS]
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TITLE: Co-Incubation protocol for transforming heterotrophic dinoflagellates (e.g. Oxyrrhis marina)
AUTHORS:  Lu Wang, Brittany Sprecher,  Huan Zhang,  and Senjie Lin

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

[STEPS]
?. [Prepare vector and transform into Escherichia coli]
Prepare a plasmid carrying proper promoter, selection marker, and reporter gene. Transform this plasmid into E. coli cells using standard heat shock protocol.
?. [Grow transformed E. coli]
Grow E. coli cells at a volume equal to 1/10 the desired volume of dinoflagellate transformation culture until the E. coli culture reaches an OD600=0.7.
?. [Harvest transformed E. coli cells]
Centrifuge the cells at 3,000g for 5 mins to pellet the E. coli.
?. [Mix transformed E. coli cells with prey alga]
Discard the supernatant and resuspend the cells to twice the volume with either the favored prey of the species (Dunaliella tertiolecta) for transformation or in L1 media seawater.
?. [Mix transformed E. coli cells and prey alga with dinoflagella]
Add the E. coli prey or seawater mix to the dinoflatellate for transformation.
?. [Co-incubation]
After incubation at normal culture condition for six hours, add LB with a volume equal to 1/10 the total volume. This step is not universal for all heterotrophic dinoflagellates so should be tested with every new species.
?. [Add fresh medium and selection agent]
After 24 hours add the same volume of new media and add the selection reagent to the media. Depending on the species it may be better to add the selection reagent after 42 hours.
?. [Add fresh medium and selection agent]
After 42 hours observe the culture under blue light.
TITLE: GYROS
AUTHORS: Anish Chhetri

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">I would absolutley go for StrainControl Laboratory Manager. I find this software extremely good and easy to use. Lets you manage strains, plasmids, oligos, antibodies and inventories.</div></div>

[STEPS]
?. [1]
Prime the instrument 200%
?. [1]
Download the sequence file
?. [1]
MRD of samples 1:2
?. [1]
Capture Prep
10 Mass/Volume Percent
?. [1]
Detector Prep
?. [1]
Open the Gyros Software
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>Background</strong></p>
<p>The use of mobile technology such as phone applications (apps) has been proposed as an efficient means of providing health and clinical information in a variety of healthcare settings. We developed the Health-e Babies app as an Android smart phone application for pregnant women attending a tertiary hospital in a low socio-economic community, with the objective of providing health information about early pregnancy that would increase maternal confidence and reduce anxiety. Based on our earlier research, this form of health communication was viewed as a preferred source of information for women of reproductive age. However, the pilot study had a poor participation rate with 76% (n=94) not completing the study requirements. These initial findings raised some very important issues in relation to the difficulties of engaging women with a pregnancy app. This paper analyses the characteristics of the participants who did not complete the study requirements in an attempt to identify potential barriers associated with the implementation of a pregnancy app.</p>
<p><strong>Methods</strong></p>
<p>This retrospective review of quantitative and qualitative data collected at the commencement of the Health-e Babies App trial, related to the participant’s communication technology use, confidence in knowing where to seek help and mental health status, maternal-fetal attachment and parenting confidence. Engagement and use of the Health-e Babies App was measured by the completion of a questionnaire about the app and downloaded data from participant’s phones. Mental health status, confidence and self-efficacy were measured by questionnaires.</p>
<p><strong>Results</strong></p>
<p>All women were similar in terms of age, race, marital status and level of education. Of the 94 women (76%) who did not complete the trial, they were significantly more anxious as indicated by State Trait Anxiety Inventory (p=0.001 Student T-test) and more likely to be unemployed (50% vs 31%, p=0.012 Student T-Test).</p>
<p><strong>Conclusion</strong></p>
<p>This study provides important information about the challenges associated with the implementation of a pregnancy app in a socially disadvantaged community. The data suggests that factors including social and mental health issues, financial constraints and technological ability can affect women’s engagement with a mobile phone app.</p>
<p><strong> </strong></p>

[STEPS]
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TITLE: BSCI:414--Lab 9: Cloning the SARS-CoV-2 Spike Protein into a E. coli     Protein Expression Vector
AUTHORS: Harley King

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The SARS-CoV-2 spike protein does not express solubly in E. coli cells. This may be due to E. coli's inability to glycosylate or create disulfide bonds. The spike protein can be cloned into a vector containing the periplasmic localization peptide (pelB) like Novagen's pET-26 so the protein is transported to the oxidizing environment of the periplasm. Disulfide bonds may also be favored by expressing in modified E. coli cells like NEB's SHuffle strain. Glycosylation may be improved by expressing in a modified E. coli cell line capable of N-linked glycosylation like CLM24. </div></div>

[STEPS]
?. [Explore the Spike Protein]
Explore the structure of the SARS-CoV-2 spike protein using Cov3D.
?. [Explore the Spike Protein]
Read abstract of Zuo et al. concerning previous attempts at expressing SARS-CoV spike protein fused with SUMO tag.
?. [Clone spike -sumo into pET28.]
Using a Gibson cloning strategy in Benchling, clone SARS-CoV-2 spike-SUMO into pET28. Open three plasmids in Benchling. Cleave all other tabs. SumoPropET-28 VectorSARS-CoV2-Spike-delta21
?. [Discuss Gibson Cloning]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol is used to dissociate adult (8-10 wk) mouse ear "on ice" (in order to reduce gene expression artifacts). The cell yield is 9000 cells/mg (213000 cells total from 23 mg tissue), with 98% viable. The protocol involves a 2-layered dissociation, incubating on ice with bacillus licheniformis enzyme. The dissociation is augmented by the use of a dounce homogenizer to help break up the tissue. 

[GUIDELINES]
Bacillus Licheniformis Enzyme Mix (1 mL per 23 mg tissue): 
100 μL b. lich 100 mg/mL (10 mg/mL final conc. - Sigma, P5380)
1 μL 0.5 M EDTA (0.5 mM final conc. - Sigma, A8806)
899 μL DPBS (no Ca, Mg) ThermoFisher (cat. #14190) 
 
Preparing enzymes: 
The enzyme is made up in DPBS (#14190). It is aliquoted and stored at -80 °C at 100 mg/mL in 100 μL aliquots.
 
Reagents
Bovine Serum Albumin - Sigma (A8806)
Hypothermosol FRS – Sigma (H4416)
Red Blood Cell Lysing Buffer – Sigma (R7757)
 
Required supplies:
2 mL dounce homogenizer – Bellco (1984-10002)
Centrifuge for 1.5 mL, 15 mL conicals 
Pipettes and pipet tips 
15 ml Conicals (MLS) 
1.5 mL tubes (MLS) 
30 µM filters - Miltenyi (130-098-458) 
Petri dishes (MLS) 
Razor blades (MLS) 
Ice bucket w/ice 
Hemocytometers - InCyto Neubauer Improved (DHC-NO1-5)

[STEPS]
SECTION: Isolating tissue
?. 
SECTION: Isolating tissue
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SECTION: 1st layer
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SECTION: 1st layer
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SECTION: 1st layer
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SECTION: 1st layer
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SECTION: 1st layer
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SECTION: 2nd layer
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SECTION: 2nd layer
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SECTION: 2nd layer
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SECTION: Preparing cells for single cell sequencing
?. 
SECTION: Preparing cells for single cell sequencing
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SECTION: Preparing cells for single cell sequencing
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SECTION: Preparing cells for single cell sequencing
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SECTION: Preparing cells for single cell sequencing
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SECTION: Preparing cells for single cell sequencing
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SECTION: Preparing cells for single cell sequencing
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TITLE: Methods and protocols from Chaiyarat et al. (2019) for systematic reintroduction of bateng (Bos javanicus)
AUTHORS: Rattanawat Chaiyarat, Namphung Youngpoy, Praeploy Kongsurakan, Seree Nakbun

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Banteng (</span><span style = "font-style:italic;">Bos javanicus</span><span>) underwent general medical checkups and received minimal human contact in 302 ha enclosure.They were train in transportation boxes(1 m × 2.5 m × 1.8 m, width × long × high)over6 months.They werekept in groups in soft release cage for 4 months. Natural food plants,fresh water and artificial salt licks were provided.Immobilizations were controlledwith anesthetic drugsby veterinarians and fitted with radio collars.The radio signals were monitored every week through ground tracking, using homing in and triangulation techniquesvia VHF signals.Cameras traps were installed,memory cards and batteries were changed every month in each location.</span></div></div>

[STEPS]
TITLE: Miraprep: Fast Plasmid Prep
AUTHORS: Zaki  Molvi

[DESCRIPTION]
An adaptation of the original miraprep protocol published by Pronobis et al. PLoS One (2016).

[STEPS]
SECTION: Introduction
1. My adaptation of the miraprep method originally published by Pronobis et al. The miraprep method is highly preferred since plasmid prep kits larger than minipreps take too long and require scarier, louder centrifuges. 
SECTION: Protocol
2. Inoculate an overnight culture in 2xYT and appropriate antibiotic1
The next day, pour confluent culture into 50 mL tubes and centrifuge at 3500rpm 4C for 10-30 min.
Decant supernatant
Resuspend each tube in 2mL Qiagen Buffer P1
To each tube, add 2mL Qiagen Buffer P2. Invert tubes several times and let sit for 3 min.
To each tube, 2mL Qiagen Buffer N3. Invert tubes several times and let sit at RT for about 5 minutes or until debris forms a distinct layer on top of the lysate
Harvest lysate from bottom layer without touching cell debris and filter through a 0.2-0.4um filter2
Add 1mL lysate to as many Qiagen spin columns as needed
Spin at max speed in tabletop centrifuge for 1 min.
Discard flowthrough, add 1mL Buffer PE to each spin column
Spin at max speed in tabletop centrifuge for 1 min.
Discard flowthrough, spin again at max speed for 2 minutes.
Place each column on an eppendorf tube
Add 100uL nuclease-free water to each column and let sit for 2-3 min.3
Spin each column at max speed for 1 min to elute plasmid.
Collect and pool eluate.
SECTION: Notes
3. 1. 2xYT is or another rich medium is preferred over LB for higher plasmid yield
2. Filtering the lysate precludes the 10 min. spin step used to clear the lysate
3. I prefer eluting in water over Tris/Buffer EB for better Sanger sequencing and electroporation results
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol is designed to purify single- or double-stranded DNA fragments from PCR and other enzymatic reactions (see page 8). For cleanup of other enzymatic reactions, follow the protocol as described for PCR samples or use the MinElute Reaction Cleanup Kit. Fragments ranging from 100 bp to 10 kb are purified from primers, nucleotides, polymerases, and salts using QIAquick spin columns in a microcentrifuge.

[BEFORE_START]
Add ethanol (96&ndash;100%) to Buffer PE before use (see bottle label for volume).<br />All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a conventional tabletop microcentrifuge at room temperature.<br />Add 1:250 volume pH indicator I to Buffer PB (i.e., add 120 &micro;l pH indicator I to 30 ml Buffer PB or add 600 &micro;l pH indicator I to 150 ml Buffer PB). The yellow color of Buffer PB with pH indicator I indicates a pH of &le;7.5.<br />Add pH indicator I to entire buffer contents. Do not add pH indicator I to buffer aliquots.<br />If the purified PCR product is to be used in sensitive microarray applications, it may be beneficial to use Buffer PB without the addition of pH indicator I.

[STEPS]
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TITLE: LRRK2 Staining in mouse brain sections 
AUTHORS: Chuyu Chen

[DESCRIPTION]
LRRK2 is a widely expressed, multifunctional protein.LRRK2 mutations are the major cause to inherited and sporadic PD. Staining of LRRK2 in mouse brain can be challenging. This protocol provides an optimised way to achieve LRRK2 staining in mouse brain sections. 

[STEPS]
1. Prepare brain cryosections in 20μm thickness
2. Blocking with 5% serum (preferably same serum as host of secondary antibody) 30 min RT
3. Wash with PBS 5 min 2x
4. Permeabilize with 0.5% Triton 15 min  RT
5. Wash with PBS 5 min 2x
6. VECTOR® antigen unmasking solution 48ul/5ml water (in small glass sample vial). Microwave 10-20sec until boiled before use (or pre-heat to 100°C on heat plate)
7. Incubate at 100°C for 30min (<5 section / 5ml) then cool to room temperature (about 20min)
8. Wash with PBS 5 min 2x
9. Blocking with 5% serum 16-24hr  4°C
10. Wash with PBS 5 min 2x
11. Primary incubation 1% serum + 0.1% Triton 30-48 hr  4°C LRRK2 1:200 (Catalog number: N241A); Co-stained often with TH antibody
12. Wash with PBS 5 min 2x
13. Secondary incubation 1% serum + 0.1% Triton 1-2 hr RT Alexa Fluor 1:300
14. Wash with PBS 5 min 2x
15. Mount with ProLong™ Gold Antifade Mountant with DAPI
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>A real-time PCR targeting the DNA-dependent RNA polymerase of Orthopoxviruses.</p>
<p>This protocol was designed and developed at this laboratory.</p>

[GUIDELINES]
<ul>
<li>If using a different brand or model of real-time thermocycler, check the concentration of ROX is adequate.</li>
<li>Method assumes the user is familiar with the thermocycler and software used to run the protocol.</li>
</ul>

[STEPS]
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TITLE: JMN-MSMP Muscle Bulk RNAseq
AUTHORS: ccherry

[DESCRIPTION]
RNA sequencing of muscle

[STEPS]
SECTION: Bulk RNA sequencing
1. cDNA synthesis and library preparation were performed using the TruSeq RNA Library Prep Kit V2 following the manufacturer's protocol. 
SECTION: Bulk RNA sequencing
2. The Agilent BioAnalyzer system was used to perform quality control on samples during the library prep process as suggested in the manufacturer's protocol and RNA samples with RIN scores below 7 post-cDNA synthesis were excluded from analysis.
SECTION: Bulk RNA sequencing
3. Libraries were pooled and sequenced to a target depth of 30M unique paired end 50bp x 50bp reads per sample using a NovaSeq 6000.
TITLE: IHC AD neuropathology protocol 
AUTHORS: Christiana Bjorkli

[DESCRIPTION]
Immunohistochemical processing was conducted on tissue from chronically implanted animals, as well as non-implanted transgenic mice for characterization of AD neuropathology.

[STEPS]
SECTION: Day 1 fluorescent IHC
1. Heat-induced antigen retrieval on all tissue at 60 °C for 2 hours in phosphate buffer  (PB).

SECTION: Day 1 fluorescent IHC
2. Wash sections 3 x 10min in PB containing 0.2 % Triton X-100  (PBT+). 
SECTION: Day 1 fluorescent IHC
3. Block sections using 5 % normal goat serum in PBT+ for 1 hour. 
SECTION: Day 1 fluorescent IHC
4. Incubate sections with the primary antibodies Iba1 (1:1000; ab15690, Abcam, Cambridge, UK), McSA1 (1:1000; MediMabs, Montreal, Canada; characterization), and Aβ42 (1:1000; IBL America, Minnesota, USA; characterization) in PBT+ for 4 hours at 4 °C.

SECTION: Day 1 DAB
5. Heat-induced antigen retrieval on all tissue at 60 °C for 2 hours in phosphate buffer  (PB).
SECTION: Day 1 DAB
6. Wash in PB for 2x 10 minutes. 
SECTION: Day 1 DAB
7. Permeabilize with 0.5 % Triton‐X‐100 in Tris‐buffered saline (TBS‐Tx; 50 mm Tris, 150 mm NaCl, pH 8.0) for 10 minutes.
SECTION: Day 1 DAB
8. Block with 10 % normal goat serum in TBS‐Tx for 30 minutes. 
SECTION: Day 1 DAB
9. Incubate with the primary antibody (AT8, 1:1000) in TBS‐Tx overnight at 4 °C

SECTION: Day 2 fluorescent IHC
10. Wash sections for 3 x 10 minutes with PBT.
SECTION: Day 2 fluorescent IHC
11. Incubate sections with Alexa Fluor 546 to visualize Iba1 and McSA1, and Alexa Fluor 488 to visualize Aβ42, for 2 hours at room temperature, protected from light.
SECTION: Day 2 fluorescent IHC
12. Wash sections for 10 minutes with 4 ′, 6-diamidino-2-fenylindol (DAPI; 1:10 000; Sigma-Aldrich, Saint-Louis, MO, USA) and PB, followed by 3 washes for 10 minutes with PB. 
SECTION: Day 2 DAB
13. Wash sections with TBS‐Tx for 3 × 10 minutes. 

SECTION: Day 2 DAB
14. Incubate with a biotinylated goat anti‐mouse secondary antibody (1:500; Sigma‐Aldrich, St Louis, MO, USA) in TBS‐Tx for 90 minutes.
SECTION: Day 2 DAB
15. Wash sections for 3 x 10 minutes with TBS‐Tx. 
SECTION: Day 2 DAB
16. Incubate with ABC (Vectastain ABC kit, Vector Laboratories, Burlingame, CA, USA) for 90 minutes.
SECTION: Day 2 DAB
17. Wash with TBS‐Tx for 3 x 10 minutes. 
SECTION: Day 2 DAB
18. Wash with Tris‐HCl for 2 × 5 minutes. 
SECTION: Day 2 DAB
19. Incubate tissue with 0.67 % diaminobenzidine (DAB) and 0.024 % H2O2 for 10 minutes before a final wash with Tris‐HCl (2 × 5 minutes).
SECTION: Mounting sections
20. Mount tissue on cut edge frosted glass slides (VWR International, Radnor, PA, USA) with Tris-HCl and leave to dry  for at least 4 hours on a 38 °C heating plate, protected from light.

SECTION: Mounting sections
21. Place mounted tissue in Xylene (VWR International, Radnor, PA, USA) for at least 5 minutes for defatting and to remove excess water from the tissue, and then coverslip with Entellan (VWR International, Radnor, PA, USA) containing Xylene.
SECTION: Mounting sections
22. Leave the mounted tissue to dry overnight, protected from light.
TITLE: Revival of S. elongatus from plates
AUTHORS: Akashdutta  

[DESCRIPTION]
On receiving wild type or tranformed S. elongatus plates, one needs to revive them for further studies. Revival of S. elongatus involves making its liquid culture in BG-11 medium. The following protocol describes the liquid culture preparation from BG-11 plates.

[STEPS]
1. Take a 50 mL  autoclaved flask.
 


2. Take a 10 mL  aliquot of BG-11 solution in a falcon tube and transfer it into the flask. Keep the flask aside
 
 

4. Dip the pipette tip in the aliquot of the BG-11 medium and gently mix.
 



3. Take the cyanobacteria plate and gently scrape it with the tip of a 1 mL loaded micropipette. 


 


5.  For the engineered strains, apply appropriate antibiotics (10μg/mL kanamycin and 4μg/mL gentamicin) in the BG-11 medium.
6. Store all the flasks in a photo incubator at 15000 lux,