eamag/protocols-with-steps huggingface dataset:
TITLE: Ligation
AUTHORS: Georgina Elisa Diego Macías, Ana Belem García González, Irán Alessandra Chaparro Rodríguez, Jair Alexis Gardea Sáenz

[DESCRIPTION]
Protocol for a Ligation with Master Mix Ligase after a restriction digestion protocol had been made.

[STEPS]
1. Add5 µL of Master Mix Ligase

2. Add the a quantity of the DNA vector digested by EcoRI and PstI in a molar ratio 1:3 with the DNA insert 
3. Add the a quantity of the DNA insert digested by EcoRI and PstI in a molar ratio 3:1 with the DNA vector
4. Add water up to 20 µL

5. Ligate for 1200 min at 22°C

6. Transform with5 µLof the product

10. 
10.
TITLE: Lentiviral_vector_production_with_PEI
AUTHORS: Kenneth Matreyek, Anna Bruchez

[STEPS]
?. [Preparation]
This protocol will require transfecting HEK 293T cells. Make sure you have some 293T cells thawed and growing for at least a couple of days before doing this protocol. Furthermore, prepare stocks of PEI and Diluent if you have not done so already.
?. [Transfection]
Prepare the mixture in a sterile microcentrifuge tube.
?. [Preparation]
PEI solution:   in H2O. I have used "Polysciences, Inc.Supplier Diversity Partner PEI MAX - Transfection Grade Linear Polyethylenimine Hydrochloride (MW 40,000)"Dissolve PEI in endotoxin-free dH2O that has been heated to approximately  .Adjust pH until it reaches  , filter sterilize (0.22 uM filter), aliquot, and store at  . A working stock can be kept at  .
[PEI]
80 °C
-20 °C
4 °C
?. [Preparation]
Diluent solution:   , For  :Get   distilled waterAdd Add
[HEPES]
[NaCl]
500 mL
445 mL
[HEPES]
15 mL
[NaCl]
?. [Transfection]
If transfecting a 10cm plate (eg. when trying to make larger stocks of lentiviral vectors):Aim for a total volume of Diluent + DNA of Add  . When using VSV-G, I do a 5:5:1 ratio packaging plasmid, transfer vector, and pMD-VSVG.Add  to the diluted DNA mixture. Immediately pipet up and down and / or vortex.Incubate between  and  Don't go over .Trypsinize, count, and plate  10 million cells per 10cm plate, in a total volume of  .
500 µl
[total DNA]
[PEI]
[(minutes)]
[(minutes).]
[(minutes)]
10 mL
?. [Change media]
The next day (between 12 and 24 hours), replace with fresh media. To increase lentivector concentration of the supernatants, use  for 10cm plates and  for 6-wells.
6 mL
1.5 mL
?. [Transfection]
If transfecting an individual 6-well (eg. when trying to test out some lentiviral vectors):Aim for a total volume of Diluent + DNA of Add  . When using VSV-G, I do a 5:5:1 ratio packaging plasmid, transfer vector, and pMD-VSVG.Add  to the diluted DNA mixture. Immediately pipet up and down and / or vortex.Incubate between  and  Don't go over .Trypsinize, count, and plate  1.5 million cells per 6-well, in a total volume of  .
80 µl
[total DNA]
[PEI]
[(minutes)]
[(minutes).]
[(minutes)]
2 mL
?. [Collect the supernatants]
Collect media over the next four days and store pooled supernatant at . If using a viral envelope protein that is able to enter HEK 293T cells (eg. VSV-G or Ebola virus GP), it may be wise to collect multiple times in one day (Once when entering the lab, and one when going home), as the viral particles will be able to re-enter the producer cells essentially becoming a dead-end product. This may not be as important for viral envelopes with receptors that are not normally expressed in 293T cells (eg. SARS CoV), although its possible that virus stored at  will still be more stable than virus left at .
4 °C
4 °C
37 °C
?. [Prepare the supernatants for use]
The supernatants may still have detached 293T cells floating which may "contaminate" downstream cultures.
?. [Prepare the supernatants for use]
Centrifuge the supernatant at greater than .Aliquot and store the lentivector supernatant at .
Centrifuge: 300 34, 3 min, 4 10
-80 °C
TITLE: Aspergillus nidulans protoplast isolation for transfections
AUTHORS: Andrew Liu, Jai Denton

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Modified </span><span style = "font-style:italic;">Aspergillus nidulans</span><span> protoplast isolation using Novozyme VinoTaste Pro as an enzyme source. Protocol from previously established method adapted to produce viable protoplasts for transfections from an inexpensive and commercially available cellulase/chitinase source.</span></div></div>

[STEPS]
?. [Grow Overnight Culutre]
Late afternoon-evening the day before, grow scraped condispores (two arms from MM complete agar plate, ~ 1 x 108 total spores) in 30ml liquid CM, supplemented with pyridoxine and riboflavin, overnight at 25°C 18-20 hrs or 30°C 11-12 hrs on orbital shaker at 150rpm. Growth can be arrested at 4°C for an hour or two prior to protoplasting if needed.
?. [Prepare Protoplast Solution]
An hour before protoplast isolation, prepare 25ml fresh 2x PP Solution in 50ml conical tube as follows: 13.7ml 2M KCL480mg Citric Acid (Anhydrous) 6.4ml 1.1 M KOH3.2g VinoTaste Pro (Novozymes) ~3.0ml ddH20 (final volume 25ml)
?. [Prepare Protoplast Solution]
Shake vigorously in 50ml conical tube. Filter sterilize through 125 or 250ml single use 0.22μm low-binding SFCA filter unit (or rinse CA filter with liquid CM prior to remove surfactant). Allow 30-40min to filter using house vacuum. During this time, harvest hyphae.
?. [Harvest Hyphae & Cell Wall Digestion]
Collect hypha over a fine mesh or sterile filter paper (or single use polyester tea-bag sprayed with 70% Ethanol).
?. [Harvest Hyphae & Cell Wall Digestion]
Using a heat sterilized spatula, wash with 10ml CM, gather hyphae and place into 3ml of CM in a 15ml conical tube; when completed, note volume.
?. [Harvest Hyphae & Cell Wall Digestion]
Add an equal volume of room temp 2x PP solution.
?. [Harvest Hyphae & Cell Wall Digestion]
?. [Isolate Protoplasts]
When satisfactorily digested, filter undigested material and hyphal clusters with 70μm MACS Smart Strainer (catalog number 130-110-916) into 15ml tube, rinse mesh and remaining residue with 2ml CM.
?. [Isolate Protoplasts]
Slowly underlay 1-2ml filter sterile 1.2M sucrose solution using a sterile 9-inch Pasteur pipette.
?. [Isolate Protoplasts]
Spin at 1800 x g for 10 min at 4°C.
?. [Isolate Protoplasts]
Collect free protoplasts into 1.5ml Eppendorf tube at the interface with sterile Pasteur pipette. Place on Ice in 2.0ml Eppendorf tube.
?. [Isolate Protoplasts]
Add cold STC (1.2M sorbitol, 10mM Tris pH7.5, 10mM CaCl2) to 1.5ml and mix by very gentle inversion 3 times, spin at 1800 x g for 10min at 4°C.
?. [Isolate Protoplasts]
Carefully remove supernatant and resuspend cell pellet in cold STC 300-500μl STC for 3-5 transfections.
?. [Isolate Protoplasts]
TITLE: Creation of low-oxygen conditions.
AUTHORS: Shengyi Yan, 宏亮 董

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>We use Na</span><span style = "vertical-align:sub;">2</span><span>SO</span><span style = "vertical-align:sub;">3 </span><span>to create low-oxygen conditions in our experiment. </span></div></div>

[STEPS]
?. [Changes of dissolved oxygen with time in the LB medium with different concentrations of Na2SO3.]
Prepare LB medium with different concentrations of Na2SO3.
?. [Changes of dissolved oxygen with time in the LB medium with different concentrations of Na2SO3.]
AB1Na2SO3(100g/L)LB medium20μL20mL320μL20mL440μL20mL5100μL20mL6200μL20mL7400μL20mL
AB1Na2SO3(100g/L)LB medium20μL20mL320μL20mL440μL20mL5100μL20mL6200μL20mL7400μL20mL
?. [Changes of dissolved oxygen with time in the LB medium with different concentrations of Na2SO3.]
Use Dissolved Oxygen Meter to measure dissolved oxygen of LB medium with different concentrations of Na2SO3  in 0h、1h、2h、5h.
?. [β-Gal enzyme activity determination—Changes of ABS with time in the LB medium with different concentrations of Na2SO3.]
Add 50ml LB medium and 200ul bacteria solution to conical flask, shake overnight at 37℃.
?. [β-Gal enzyme activity determination—Changes of ABS with time in the LB medium with different concentrations of Na2SO3.]
Take 5 ml in 5 50ml centrifuge tubes separately, centrifuge the bacteria at 3000xg at room temperate for 5 min.Discard the supernatant.
?. [β-Gal enzyme activity determination—Changes of ABS with time in the LB medium with different concentrations of Na2SO3.]
Prepare LB medium with different concentrations of Na2SO3.dd Na2SO3 to LB medium.  AB1Na2SO3(100g/L)LB medium20ul20ml3100ul20ml4200ul20ml5400ul20ml6800ul20ml
AB1Na2SO3(100g/L)LB medium20ul20ml3100ul20ml4200ul20ml5400ul20ml6800ul20ml
?. [β-Gal enzyme activity determination—Changes of ABS with time in the LB medium with different concentrations of Na2SO3.]
Add LB medium with different concentrations of Na2SO3 to 5 50ml centrifuge tubes separately.
?. [β-Gal enzyme activity determination—Changes of ABS with time in the LB medium with different concentrations of Na2SO3.]
Add 2ul IPTG to 5 50ml centrifuge tubes separately,and reascend.  Shake 5h at 37℃.
?. [β-Gal enzyme activity determination—Changes of ABS with time in the LB medium with different concentrations of Na2SO3.]
Use Microplate Reader to measure ABS with different concentrations of Na2SO3  in 0 h、1h、2h、5h.
TITLE: 10x Protocols: Chromium Single Cell/Nuclei Gene Expression Flex Multiplex -- University of Minnesota TMCs (CG000527 Rev D)
AUTHORS: IOx Genomics, Laura Niedernhofer

[DESCRIPTION]
10x Genomics Chromium Single Cell Expression flex protocol for library construction. 


Protocol ID# (CG) and Revision letter provided here: 
    10x protocol CG000527, Revision D -- Library construction of 10x fixed single cell or nuclei


Note: These protocols may not be the current version offered by the company but were used to produce the specific datasets connected to them. Please review the company support websites for the most recent versions of the protocols prior to starting your experiment. 

[STEPS]
1. https://www.10xgenomics.com/products/single-cell-gene-expression-flex
https://www.10xgenomics.com/support/single-cell-gene-expression-flex
2. Complete single cell or nuclei isolation and 10x fixation prior to starting this protocol
3.
TITLE: RNA Isolation from Plant Tissue Protocol 11: Tri Reagent Method
AUTHORS: 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Implemented by: Michael Melkonian and Barbara Surek</div><div class = "text-block"><span>The protocol for RNA isolation using </span><a href="https://www.protocols.io/view/protocol-11-tri-reagent-method-4ydgxs6/materials" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">TRI Reagent</span></a><span> (Molecular Research Center, Cincinnati OH) is analogous to the TRIzol LS Reagent protocol except that 1 ml of TRI Reagent is added to each 250 μl of homogenized material (= approx. 50–100 mg packed cell volume) (step 3, </span><a href="https://www.protocols.io/view/protocol-10-trizol-ls-reagent-method-4rwgv7e" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Protocol 10</span></a><span>). </span></div><div class = "text-block">For background, TRI Reagent combines phenol and guanidine thiocyanate in a monophasic solution to inhibit RNase activity during RNA isolation.</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]
TITLE: Metabolomics and Lipidomics Sample Preparation
AUTHORS: Kevin Contrepois

[DESCRIPTION]
Scope:
To describe the procedure to extract metabolites and complex lipids using a biphasic separation with cold methyl tert-butyl ether (MTBE), methanol and water.

Expected outcome/data:
Metabolites and complex lipids are prepared to be analyzed usin untargeted metabolomics and targeted lipidomics approaches, respectively.

[STEPS]
1. Roughly 30 mg of frozen tissues were homogenized in 500 µl ice-cold methanol by bead beating (MP bioscience cat# 6913-100, Solon, OH) at 4°C (2 x 45 s). 
2. 1 ml of MTBE was added to 300 μl of the homogenate spiked-in with 40 µL deuterated lipid internal standards (Sciex, cat#: 5040156, lot#: LPISTDKIT-101). 
3. The samples were then sonicated (3 x 30 s) and agitated at 4°C for 30 min.
4. After addition of 250 μl of ice-cold water, the samples were vortexed for 1 min and centrifuged at 14,000 g for 5 min at 20°C. 
5. The upper organic phase contains the lipids, the lower aqueous phase contains the metabolites and the proteins are precipitated at the bottom of the tube. For quality controls, 3 reference plasma samples (40 µL plasma) and 1 preparation blank were prepared in parallel.
SECTION: Metabolites
6. Proteins were further precipitated by adding 700 μl of 33/33/33 acetone/acetonitrile/methanol spiked-in with 15 labeled metabolite internal standards to 300 μl of the aqueous phase and 200 μl of the lipid phase and incubating the samples overnight at -20°C
SECTION: Metabolites
7. After centrifugation at 17,000 g for 10 min at 4°C, the metabolic extracts were dried down to completion under a stream of nitrogen and resuspended in 100 μl 50/50 methanol/water.
SECTION: Complex lipids
8. 700 µl of the organic upper phase was dried down and resolubilized in 200 μl of methanol for storage at -20°C until analysis. 
SECTION: Complex lipids
9. The day of the analysis, samples were dried down, resuspended in 300 μl of 10 mM ammonium acetate in 90/10 methanol/toluene and centrifuged at 16,000 g for 5 min at 24°C.
TITLE: Impact of glucose-6-phosphate dehydrogenase deficiency on dengue infection in Myanmar childrened protocol
AUTHORS: Win Lai May, Myat Phone Kyaw, Stuart D. Blacksell, Sasithon Pukrittayakamee, Kesinee Chotivanich, Borimas Hanboonkunupakarn, Khin Nyo Thein, Chae Seung Lim, Janjira Thaipadungpanit, Thomas Althaus, podjanee jittamala

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Glucose-6-phosphate dehydrogenase (G6PD) deficiency may affect the clinical presentation of dengue due to the altered redox state in immune cells. We aimed to determine the association between G6PD deficiency and severity of dengue infection in paediatric patients in Myanmar. A cross-sectional study was conducted among paediatric patients aged 2–13 years with dengue in Yankin Children Hospital, Myanmar. One hundred and ninety-six patients positive for dengue infection, as determined via PCR or ELISA, were enrolled. Dengue severity was determined according to the 2009 WHO classification guidelines. Spectrophotometric assays determined G6PD levels. The adjusted median G6PD value of males in the study population was used to define various cut-off points according to the WHO classification guidelines. G6PD genotyping for Mahidol, Kaiping and Mediterranean mutations was done for 128 out of 196 samples by real-time multiplex PCR. 51 out 196 patients (26.0%) had severe dengue. The prevalence of G6PD phenotype deficiency (< 60% activity) in paediatric patients was 14.8% (29/196), specifically, 13.6% (14/103) in males and 16.2% (15/93) in females. Severe deficiency (< 10% activity) accounted for 7.1% (14/196) of our cohort, occurring 11.7% (12/103) in males and 2.2% (2/93) in females. Among 128 samples genotyped, the G6PD gene mutations were detected in 19.5% (25/128) of patients, with 20.3% (13/ 64) in males and 18.8% (12/64) in females. The G6PD Mahidol mutation was 96% (24/25) while the G6PD Kaiping mutation was 4% (1/25). Severe dengue was not associated with G6PD enzyme deficiency or presence of the G6PD gene mutation. Thus, no association between G6PD deficiency and dengue severity could be detected.</div></div>

[STEPS]
TITLE: Identification chart for common Daphnia species in lakes in Michigan, and their most common parasites
AUTHORS: Meghan Duffy, Kristel Sanchez

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a guide to the common Daphnia (& one Ceriodaphnia) species found in stratified lakes in Michigan, as well as the most common parasites that infect Daphnia in those lakes. This is not a formal key, but is a resource that was developed by members of the Duffy Lab (especially Kristel Sánchez and Camden Gowler) to assist lab members as they count field samples. </div></div>

[STEPS]
TITLE: Monosynaptic Rabies Tracing
AUTHORS: Alexandra Nelson, Michael Ryan

[DESCRIPTION]
This protocol describes the steps for performing stereotaxic surgery in mice. It is applicable to intracranial injections (e.g. virus, drug) and placement of implants (e.g. optical fibers, electrode arrays) into targeted regions of mouse brains.

[STEPS]
1. Rabies injections were performed in a BSL-2 surgical suite following the protocol described in dx.doi.org/10.17504/protocols.io.n2bvj6qynlk5/v1
1.1. For modified, G-deleted rabies viruses, first inject a Cre-dependent helper virus (AAV-DIO-sTpEpB-GFP) to restrict expression of the EnvA receptor (TVA) and rabies glycoprotein necessary for subsequent rabies virus infection.
1.2. 2 weeks later, inject the modified, G-deleted rabies virus (EnvA-G-deleted-rabies-mCherry) into the same coordinates as the helper virus.
1.3. For a detailed protocol regarding subsequent perfusion, sectioning, staining, imaging, and analysis with MBF NeuroInfo software, see Eastwood, et al., 2019 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6570587/
TITLE: Copy of Immunohistochemistry
AUTHORS: Ning Zhang, Yu Kuang

[STEPS]
?. Deparaffinize/hydrate sections: a.Incubate sections in three washes of xylene for 5 minutes each.b.Incubate sections in two washes of 100% ethanol for 10 minutes each.c.Incubate sections in two washes of 95% ethanol for 10 minutes each.
?. Wash sections twice in dH2O for 5 minutes each.
?. Incubate sections in 3% hydrogen peroxide for 10 minutes.
?. Wash sections in wash buffer for 5 minutes.
?. Block each section with 100-400 µl blocking solution for 1 hour at room temperature.
?. Remove blocking solution and add 100-400 µl primary antibody diluted in recommended antibody diluent to each section. Incubate overnight at 4°C.
?. Remove antibody solution and wash sections in wash buffer three times for 5 minutes each.
?. Add 100-400 µl biotinylated secondary antibody, diluted in TBST per manufacturer’s recommendation, to each section. Incubate 30 minutes at room temperature.
?. If using ABC avidin/biotin method, prepare ABC reagent according to the manufacturer’s instructions and incubate solution for 30 minutes at room temperature.
?. Remove secondary antibody solution and wash sections three times with wash buffer for 5 minutes each.
?. Add 100-400 µl ABC reagent to each section and incubate for 30 minutes at room temperature.
?. Remove ABC reagent and wash sections three times in wash buffer for 5 minutes each.
?. Add 100-400 µl DAB or suitable substrate to each section and monitor staining closely.
?. As soon as the sections develop, immerse slides in dH2O.
?. If desired, counterstain sections in hematoxylin per manufacturer’s instructions.
?. Wash sections in dH2O two times for 5 minutes each.
?. Dehydrate sections: a.Incubate sections in 95% ethanol two times for 10 seconds each.b.Repeat in 100% ethanol, incubating sections two times for 10 seconds each.c.Repeat in xylene, incubating sections two times for 10 seconds each.
?. Mount coverslips.
TITLE: Purification and Crystallization of ATG9 HDIR-ATG101:ATG13 complex
AUTHORS: Adam   Yokom, Xuefeng Ren

[DESCRIPTION]
Purification and Crystallization of ATG9 HDIR (828-839) fused ATG101 (1-198):ATG13 (1-197)

[STEPS]
SECTION: Crystallization
19. ATG9 HDIR-ATG101:ATG13 complex at  6 mg/ml in 25 mM HEPES pH 7.5, 150mM NaCl, 1mM MgCl2, 1mM TCEP was used as the protein stock
SECTION: Crystallization
20. Crystallization was carried out by sitting-drop vapor diffusion using an automated liquid-handling system (Mosquito, TTP LabTech, UK) at 288 K in 96-well plates
SECTION: Crystallization
21. The protein solution was mixed with the reservoir buffer composed of 0.1 M HEPES pH 7.5, 0.2 M NaCl, 12% PEG8000 with a ratio of 1:1
SECTION: Crystallization
22. Crystal trays were checked daily using a light microscopy for crystal growth
SECTION: Crystallization
23. Large crystals were obtained in 2–4 days. Crystals were cryo-protectedin 28% glycerol/reservoir buffer and frozen in liquid N2
SECTION: Purification
7. Resuspended pellet in lysis buffer (25 mM HEPES pH 7.5, 200 mM NaCl, 2 mM MgCl2, 1 mM TCEP, 5 mM EDTA, 10% Glycerol) with 1% Triton X-100 and protease inhibitor cocktail (Thermo Scientific, Waltham, MA) 
SECTION: Expression
5. Harvest Cells 500 rpm, 10 min, 4 °C 
SECTION: Expression
4. Add mixture to cells. Let cells grow for 2880 min 
SECTION: Expression
3. Add PEI to DNA dilution. Incubate mixture for 30 min  at 37 °C 
SECTION: Expression
2. Dilute PEI with Warm Hybridoma-SFM(1X), In a separate tube, dilute DNA with Hybridoma-SFM(1X)
SECTION: Expression
1. Transfect HEK GNTI cells at concentration of 2 × 106 cells/ml
SECTION: Expression
6. Wash pellet with cold PBS. Store pellet at -80C until purification or lyse immediately 
SECTION: Purification
11. Rock supernatant with equilibrated resin for 60 min at 4 °C
SECTION: Purification
10. Load supernatant onto GST Sepharose resin using a gravity column setup
SECTION: Purification
9. Wash GST Sepharose 4B resin into lysis buffer (without Triton)
SECTION: Purification
8. Clarify lysate for 17000 rpm, 35 min, 4 °C
SECTION: Purification
12. Wash with 5CV lysis buffer (25 mM HEPES pH 7.5, 200 mM NaCl, 2 mM MgCl2, 1 mM TCEP, 5 mM EDTA, 10% Glycerol)
SECTION: Purification
13. Elute with lysis buffer plus 25 mM glutathione for GST resin
SECTION: Purification
14. Add purified His6-TEV protein to cleave GST tag at 4°C overnight
SECTION: Purification
15. Dilute 5x into SP Buffer buffer A: 30 mM MES pH 6.0, 3 mM beta-mercaptoethanol
SECTION: Purification
16. Load sample onto a HiTrap SP HP 5 ml column (GE healthcare, Piscataway, NJ)
SECTION: Purification
17. Elute from the SP column on a 70 ml linear gradient from 0–500 mM NaCl in SP buffer A. The cleavage sample was eluted at the buffer conductivity of ~ 25 mS/cm.
SECTION: Purification
18. After each fraction was analyzed by SDS gel. Fractions containing ATG9 HDIR-ATG101:ATG13 were pooled concentrated in Amicon Ultra15 concentrator (MilliporeSigma, Burlington, MA) and exchanged into 25 mM HEPES pH 7.5, 150mM NaCl, 1mM MgCl2, 1mM TCEP for crystallization.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol results in the isolation of single nucleated zoospores from a culture of Aurantiochytrium limacinum.  The point is to recover single nucleus colonies rather than single cell colonies (which may contain varying nuclei - particularly following transformation).

[STEPS]
SECTION: Prepare Culture
?. 
SECTION: Filter Culture
?. 
SECTION: Plate Zoospores
?.
TITLE: No Title
AUTHORS: 

[BEFORE_START]
<p>-Sterile polypropylene tubes (15 or 50 ml); sterile Oak Ridge-tubes</p>
<p> </p>
<p>-Steel forceps </p>
<p> </p>
<p>-Scissors</p>
<p> </p>
<p>-glass rod with rounded end</p>
<p> </p>
<p>-95% Ethanol for sterilization</p>
<p> </p>
<p>-Sterile lysis buffer (10 mM Tris, 100 mM NaCl, 1 mM EDTA, pH9)</p>
<p> </p>
<p>-Stock solutions</p>
<p>   -lysozyme (100 mg/ml)</p>
<p>   -proteinase K (20 mg/ml)</p>
<p>   -10% SDS</p>
<p> </p>
<p>-Incubator/water bath for +37ºC and +50ºC incubations</p>
<p> </p>
<p>-Glass pipettes (10 ml)+ rubber bulb</p>
<p> </p>
<p>-Saturated phenol, pH 8</p>
<p> </p>
<p>-Chloroform-isoamyl alcohol (24:1)</p>
<p> </p>
<p>-Absolute and 70% ethanol</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
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?. 
?. 
?. 
?. 
?.
TITLE: Test protocol II
AUTHORS: Abby Moore

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a test protocol</div></div>

[STEPS]
?. Use 80:20 MeOH:H2O for this step. This is not easy to access by machine.
?. If you haven't already, make a solution with the following components:
?. If you haven't already, make a solution with the following components:
?. Use this piece of equipment:
?.