eamag/protocols-with-steps huggingface dataset:
TITLE: Microwave digestion for microbes
AUTHORS: An.Huang

[DESCRIPTION]
Before conducting inductively coupled plasma mass spectrometry (ICP-MS), all samples containing organic compounds need to be digested in order to eliminate all organic parts. Microwave digestion is a commonly used method for organism digestion. This protocol describes how to conduct microwave digestion on microbes.

[STEPS]
SECTION: Preparation of digestion vessels
1. Pick out digestion vessels from preservation boxes containing nitric acid (4%). Rinse and wash the vessels thoroughly. Also wash coordinated number of caps and plugs.
 

 

SECTION: Preparation of digestion vessels
2. Put all vessels inside the oven and dried at 60 °C for at least 60 min.

SECTION: Preparation of digestion vessels
3. Take all vessels out from the oven when they are completely dried and put all vessels into the fume hood.
SECTION: Microwave digestion
5. Add samples into digestion vessels.
 

SECTION: Microwave digestion
4. Label all vessels according to the samples.
SECTION: Microwave digestion
6. Add 5 mL nitric acid (70%) and 2 mL hydrogen peroxide into the digestion vessels.
 

 


SECTION: Microwave digestion
7. Plug the vessels and screw the caps tightly.
SECTION: Microwave digestion
8. Put all vessels into microwave digester according to the manual of the digestion system.
SECTION: Microwave digestion
9. Set the digest programme according to the vessel number. Please read the digestion system manual for more instruction.
SECTION: Microwave digestion
10. When the digestion programme has finished, wait for around half an hour to let vessels cool down.
SECTION: Volume consistency
11. Open the caps and unplug the vessels when the vessels have not completely cooled to room temperature. Pour the remaining liquid inside the vessel into a 50 mL centrifuge tube.
 


 


SECTION: Volume consistency
12. Use a dropper to add 3 mL ddH2O into one vessel by the inside wall. Shake the vessel generally and pour the water into the same 50mL centrifuge tube in Step#11. Repeat this step for 6 to 8 times.
 


SECTION: Volume consistency
13. Add ddH2O until the liquid volume inside the 50 mL centrifuge tube reeds 50 mL.
 


SECTION: Volume consistency
14. The digested samples in 50 mL tubes need to be stored at 4 °C for further analysis.
SECTION: Digestion vessel storage
15. Wash all digestion vessels using ddH2O for three times.
SECTION: Digestion vessel storage
16. Submerge all vessels into nitric acid (4%) inside the preservation box.
SECTION: Microwave digestion
7.1. Here we skipped a step of pre-digestion, which is to heat these capped vessels at 80℃ overnight. Since our samples only contain microbes like bacteria, it is not a necessity to conduct this step. This part is designed for larger organisms like cellulose, which is hard to digest.
TITLE: Pythium Zoospore Production Soaking Solution
AUTHORS: Nimalka Weerasuriya

[DESCRIPTION]
Creation and test of soaking solutions to be used for large-scale zoospore production for Pythium myriotylum. This is modified from methods in: 
Nyochembeng, L. M., Pacumbaba, R. P., & Beyl, C. A. (2002). Calcium Enhanced Zoospore Production of Pythium myriotylum in vitro. Journal of Phytopathology, 150(7), 396–398. https://doi.org/10.1046/J.1439-0434.2002.00759.X

[STEPS]
SECTION: Soaking Solutions
2. Make Soaking Solutions 1, 2, 3, and Control.
Prep 4 x 1 L autoclavable bottles for each Soaking Solutions (1-3) and Control.

 

SECTION: Preparation
1. Have mature colonies of verified Pythium myriotylum growing on CMA or 1.5-2% WA 90 mm plates. Colony maturity ~7-14 days, with visible oospores.
 

 

SECTION: Soaking Solutions
2.1. Soaking Solution #1, 0.01 Molarity (m) (1 L):
1 L  at pH 7 in 1 L beaker
Add 1 g 
Filter through Whatman #1 filter paper using a funnel into a 1 L bottle.
Add a stir bar to the bottle. 
Adjust pH from pH 8.5  to pH 10.5 to pH 7.0 with  5 N  and  1 2635 to duplicate the soaking solution in original methods. 
 

SECTION: Soaking Solutions
2.2. Soaking Solution #2, 0.01 Molarity (m)+0.001 Molarity (m) (500 mL):
Transfer 500 mL of SS#1 into a clean 500 mL bottle labeled SS #2.
Add 171 mg to make 0.001 Molarity (m) solution.

 
Check that pH is pH 7.
SECTION: Soaking Solutions
2.3. Soaking Solution #3, 0.001 Molarity (m) (500 mL):
Add 171 mg to make 0.001 Molarity (m) solution in 500 mL  
Check that pH is pH 7.

SECTION: Soaking Solutions
3. Autoclave for 20 minutes on liquid cycle. Remove from autoclave immediately to reduce water evaporation and concentration changes.
 

SECTION: Testing Zoospore Production
4. Take 30 mL of each Soaking Solution   onto surface of Pythium myriotylum plate cultures that have actively produced oospores.

SECTION: Testing Zoospore Production
5. Incubate under light at Room temperature for 1440 min. 
Check for abundant sporangia that will appear after immersion.

SECTION: Testing Zoospore Production
6. After 1.5 up to 4 h every 30 minutes:
Take 80 µL from each immersion to a microcentrifuge tube with 20 µL. Gently invert to stain (or gently vortex) and immobilize zoospores.

 

SECTION: Testing Zoospore Production
7. Take 10 µL of liquid into haemocytometer and examine under 40x to quantify.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Electroporation protocol

Weinstock paper:
Matthew T Weinstock, Eric D Hesek,Christopher M Wilson, Daniel G Gibson
Vibrio natriegens as a fast-growing host for molecular biology
Nature Methods volume 13, pages 849–851 (2016)
 
 
To prepare before:
 
recovery medium
(BHI + v2 salts (204 mM NaCl, 4.2 mM KCl, 23.14mM MgCl2), and 680 mM sucrose) sterile filtration  
* i have a box of aliquots in the freezer and pre heat them before use
 
-A vial of competent cells is retrieved from storage at −80 °C and allowed to thaw on ice.
-Plasmid DNA and electrocompetent cells are combined and gently mixed in a chilled  
 1.5-mL microcentrifuge tube. 
-The cell–DNA suspension is transferred to a chilledelectroporation cuvette with a 
 0.1-cm gap size.
-Cells are electroporated with 900V (in our Electroportor we cant set other parameters ) 
* in the Weinstock paper they recommend depending on the strain 700-900V, 25 μF and  200 Ω
 
-Cells are immediatelyrecovered in 500 μL preheated  (50°C) recovery medium and  
 transferred to a 1,5-mL tube. 
 * we preheat the media to 50°C because the recovery media is cooled down by pipetting and up taking the chilled cells from the cold cuvettes
 
-The cells are recovered by incubating at 37 °C for 1.5h. 
 ( also put the agar plates for preheating in the incubator at 37°C )
-The cells are centrifuged down for one mintute at 3000g. The supernant is then discanted.
- the pellet is resuspendet in the leftover oft he media and plated out on
 warm agar plates containing appropriate antibiotic. 
* for Chloramphenicol 2µg/mL ; for Kanamycine 200µg/mL; for Carbenicillin 200µg/mL
 
-The plates are incubated for several hours or overnight at 37 °C for colonies to appear.
 

[STEPS]
TITLE: Protocol for Differentiation of Blood-Brain Barrier Endothelial Cells from Human Pluripotent Stem Cells
AUTHORS: Ethan Lippmann, Hannah Wilson, Emma Neal

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Human induced pluripotent stem cell (iPSC)-derived developmental lineages are key tools for in vitro mechanistic interrogations, drug discovery, and disease modeling. iPSCs have previously been differentiated to endothelial cells with blood-brain barrier (BBB) properties, as defined by high transendothelial electrical resistance (TEER), low passive permeability, and active transporter functions. Typical protocols use undefined components, which impart unacceptable variability on the differentiation process. We demonstrate that replacement of serum with fully defined components, from common medium supplements to a simple mixture of insulin, transferrin, and selenium, yields BBB endothelium with TEER in the range of 2,000-8,000 Ω × cm</span><span style = "vertical-align:super;">2</span><span>across multiple iPSC lines, with appropriate marker expression and active transporters. The use of a fully defined medium vastly improves the consistency of differentiation, and co-culture of BBB endothelium with iPSC-derived astrocytes produces a robust in vitro neurovascular model. This defined differentiation scheme should broadly enable the use of human BBB endothelium for diverse applications.</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/public/1618c1a37f153002e3885d7ebd72cd0a4e653895bbeb80a8934c28142d836cb2/bspy7vcw.png" /></div></div>

[STEPS]
?. [BBB differentiation (Day 0–4)]
On day 0, aspirate E8 medium and add  of E6 per well.
Note: Cells are seeded for differentiation in E8 medium according to the standardized single cell seeding protocol
2 ml
?. [BBB differentiation (Day 0–4)]
Change medium every day using  of E6 per well.
2 ml
?. [BBB expansion (Day 4–6)]
At day 4 of E6 treatment, aspirate and add  of EC medium with bFGF (basic fibroblast growth factor) and  RA to each well.
2 ml
Medium is NOT changed during expansion phase.
?. [BBB expansion (Day 4–6)]
BBB subculturing:On day 6, subculture BBB onto plates and Transwell filters according to the following protocol:.
TITLE: Flow cytometry for TH-positive iPSC derived neurons
AUTHORS: Ellen Hertz, Ellen Hertz, Gani Perez, Martha Kirby, Stacie Anderson, Yu Chen, Ellen Sidransky

[DESCRIPTION]
This protocol details the flow cytometry procedure.

[STEPS]
SECTION: Flow cytometry dopaminergic fraction
1. Wash the neurons in PBS and incubate with Membrite prestain solution, dilute 1:1000 in HBSS, for 5 min at 37 °C.
SECTION: Flow cytometry dopaminergic fraction
2. Change the prestain solution to staining solution and incubate for another 5 min at 37 °C.

SECTION: Flow cytometry dopaminergic fraction
3. Dissociate the cells with prewarmed papain with DNAse, Y-27632 and Emricasan for 10 min at 37 °C.
SECTION: Flow cytometry dopaminergic fraction
4. Quench the reaction in DMEM/Glutamax with 10% fetal bovine serum and collect by centrifugation at 400 x g, 5 min.

SECTION: Flow cytometry dopaminergic fraction
5. Resuspend the pellet in 4% paraformaldehyde and incubate for 10 min at Room temperature on a rolling wheel.

SECTION: Flow cytometry dopaminergic fraction
6. Resuspend the cells in DMEM with 10% FBS and spin at 500 x g, 5 min after which the cells were incubated in permeabilization buffer (1% BSA, 0.1% saponin in PBS, 10% normal donkey serum  for 30 min at Room temperature and thereafter label with tyrosine hydroxylase antibody or isotype controls (1:400, Cat.# P40101-150, Pel-Freeze and Cat.# AB-105-C R&D respectively) 30 min at 4 °C.

SECTION: Flow cytometry dopaminergic fraction
7. Wash the cells twice with 2% BSA and 1.5 millimolar (mM) EDTA in PBS and then incubate with diluted Alexa647-conjugated secondary antibody, for 60 min at Room temperature.

SECTION: Flow cytometry dopaminergic fraction
8. Wash the cells as described above with NucBlue Fixed Cell ReadyProbes Reagent in the second wash and resuspend in wash buffer for flow cytometry analysis.
SECTION: Flow cytometry dopaminergic fraction
9. Initial gate: Include Membrite+ cells
Second gate: Include single cell DAPI (exclude duplicates).
Third gate:  Alexa647+
TITLE: Proteolytic Peptide Desalting with C18 Hydrophilic–Lipophilic Balance (HLB) Cartridges
AUTHORS: J Bons, J P Rose, M A Watson, B Schilling

[DESCRIPTION]
Desalting of proteolytic peptide elution using C18 Hydrophilic–Lipophilic Balance (HLB) Cartridges in preparation for downstream proteomic profiling.




[STEPS]
1. Centrifuge the samples at 1,850 x g for 5 minutes at room temperature to pellet insoluble material.
2. Desalt the samples using Oasis HLB solid-phase extraction cartridges placed on top of a vacuum manifold as follows:
2.1. Condition each cartridge two times with 800 µL of the condition buffer (50% ACN, 0.2% FA).
2.2. Equilibrate each cartridge three times with 800 µL of the equilibration buffer (0.2% FA).
2.3. Load the peptide samples.
2.4. Wash each cartridge three times with 800 µL of the wash buffer (0.2% FA). 
2.5. Elute peptides with 800 µL of the elution buffer (50% ACN, 0.2% FA) followed by a further 400 µL of the same elution buffer.
3. Vacuum dry the eluted peptide solution in a centrifugal vacuum concentrator.
4. Reconstitute the dried peptides in 0.2% FA for a final protein concentration of 1 µg/µL and thoroughly mix the solution. 
5. Centrifuge at 12,000 x g at room temperature for min, and store at -20°C.
TITLE: No Title
AUTHORS: 

[BEFORE_START]
<p>Before starting, make 10X sterile Daigo solution in artificial seawater (ASW):</p>
<p>Dissolve 2.56 g Daigo powder in 1L ASW and filter sterilize through a 0.22 µm filter.</p>

[GUIDELINES]
<p> </p>
<p> </p>

[STEPS]
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TITLE: Western blot protocol for detecting ATP10B in mouse/rat brain
AUTHORS: María Sanchiz Calvo, eduard.bentea , Veerle Baekelandt

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for detection of ATP10B in rat and mouse brain tissue by Western blotting</div></div>

[STEPS]
?. [Day 1]
Prepare samples for Western blotting : proteins in  of volume (adjust with milliQ water) of 6X SDS buffer + 10% of β-mercaptoethanol
30 µg
12 µl
2.4 µl
?. [Day 1]
Boil the samples for  at
98 °C
?. [Day 1]
Load samples, together with  of mass marker (PageRuler™ Plus Prestained Protein Ladder) on a 3-8% NuPAGE™ Tris-Acetate gel. Use NuPAGE™ Tris-Acetate SDS Running Buffer for migration.
7 µl
?. [Day 1]
Run the gel at 80V for
?. [Day 1]
Run the gel at 150V for another
?. [Day 1]
Transfer the proteins on PVDF membrane (Trans-Blot® TurboTM PVDF Membrane) using Trans-Blot Turbo Transfer System (Bio-Rad), using the pre-programmed protocol STANDARD SD: , up to 1.0 A, 25 V.
?. [Day 1]
Block membranes for  using 5% milk dissolved in PBS-T 0,1% at
0 Room temperature
?. [Day 1]
Incubate membranes  in ATP10B HPA034574 (Sigma) diluted 1/500 in 5% milk PBS-T 0,1% at
4 °C
?. [Day 2]
Wash membranes with PBS-T 0,1% for 10 minutes, 5 times at  :PBS-T PBS-T PBS-T PBS-T PBS-T
0 Room temperature
?. [Day 2]
Incubate membranes for  in secondary antibody solution Goat anti-rabbit HRP (Dako) diluted 1/10000 in 5% milk PBS-T 0,1% at
0 Room temperature
?. [Day 2]
Wash membranes with PBS-T 0,1% for 10 minutes, 5 times at  :PBS-T PBS-T PBS-T PBS-T PBS-T
0 Room temperature
?. [Day 2]
Develop membranes using ECL Prime (GE Healthcare) for at least
?. [Day 1]
Vortex samples and spin down
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<strong>Materials:</strong><br /><br />1.) host and viral DNAs<br />2.) 1X TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA)<br />3.) 1 M NaOH<br />4.) 1 M Acetic acid<br />5.) nylon membrane, cut to 12.3 X16 cm (2 sheets)<br />6.) Whatman 3MM filter paper, cut 12.3X16 cm (2 sheets)<br />7.) SSC solutions: 1X, 5X, 20X<br />       20X SSC (1000 mL):
<ul>
<li>175.3 g NaCl</li>
<li>88.2 g sodium citrate</li>
<li>adjust pH to 7.0 with NaOH</li>
</ul>
8.) 96-well assay plates<br />9.) hybridot dot blot apparatus and pump<br /><br />Note: Load the membranes so that when scanned, the second blot of the set may be placed under the first, i.e., load as the following diagram shows:<br /><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/cvdc2xe.png" data-src="https://s3.amazonaws.com/pr-journal/cvdc2xe.png" data-ofn="dot blot image.png" />

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Stains zebrafish larvae with Sudan Black B, which marks neutrophil granules.</p>

[BEFORE_START]
<p>You will need a rocker/nutator/rotator at 4ºC and at RT.</p>
<p> </p>
<p>Make both 1x and 10x stocks of PBS from powder packets, in water.</p>
<p>Make 70% Ethanol solution.</p>
<p>Make PBS-Tween = 1x PBS + 0.1% (v/v) Tween20.</p>
<p>Make 10% (w/v) Potassium hydroxide (KOH) in water.</p>
<p> </p>
<p>Make Sudan Black B stock and working solution:</p>
<p>Stock: 0.18% (w/v) Sudan Black B in 70% Ethanol (store at RT, good for 1+ yr)</p>
<p>Working solution: 10 ml stock + 40 ml 70% Ethanol + 50 μl Phenol (store at RT, good for 6+ mo)</p>
<p> </p>
<p> </p>

[STEPS]
?. 
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TITLE: Natural killer cell depletion in vivo (mouse)
AUTHORS: Goran  Tomic

[DESCRIPTION]
This protocol describes a validated procedure for antibody depletion of natural killer cells in mice (C57Bl/6J and 129SvEv strains), combined with the injection of cancer cells i.v. It is based on combining a few references and testing the optimal antibody concentration and frequency of injection to achieve good level of depletion, minimise animal injection frequency, and reagent consumption. 



[BEFORE_START]
Before starting the experiment, validate the level od depletion of your batch of antibody by FACS (use spleen or blood sample to check the number of NK cells  4 or 7 days after antibody administration). Make sure to use a staining antibody for a marker other than the one targeted with a depletion antibody. I use Nkp46. 

My protocol is based on depleting the NK cells for 4 days (two doses) before injecting tumour cells, followed by a weekly administration of the depleting antibody afterwards. 

This schedule should be checked and adapted for any other setting/experiment. 

For NK cell depletion in C57Bl/6 mice, use the following antibody and corresponding isotype control:

Isotype control is Mouse IgG2a, κ (InVivoMAb, clone C1.18.4, catalog number BE0085)
NK cell neutralising antibody is Nk1.1 (Clone PK136, generated in-house from a hybridoma line, or purchased commercially)

Most references use 200-300 ug of NK1.1 antibody per mouse, with various frequencies, from 1x week to 3x week

Smyth et al. 1998 (200 ug)
Nishikado et al. 2011 (300 ug)
Victorino et al. 2015 (200 ug)

For NK cell depletion in 129SvEv mice, use the following antibody, because 129S strain NK cells do not express Nk1.1:

Polyclonal rabbit IgG (InVivoMAb, BE0095, Isotype Control)
Asialo GM1 Polyclonal Antibody, Functional Grade, eBioscience™ (ThermoFisher, Catalog # 16-6507-39)

Dose:
10 uL (anti-asialo GM1), diluted to 100 uL with PBS prior to the injection

N.B. Anti-asialo GM1 antibody also depletes basophils.

[STEPS]
1. 
Mice will receive an i.p. injection of the antibody in 200 µl PBS (Day 1) e.g. Monday
2. Mice will receive an i.p. injection of the antibody in 200 µl PBS (Day 4) e.g. Thursday
3. Mice will receive tail vein injection of the cell suspension in 100 µl PBS (Day 5) e.g. Friday
4. Mice will receive an i.p. injection of the antibody in 200 µl PBS (Day 11, 18, 25) i.e. once per week
5. Mice will be culled after 28 days from the day of tumour cell injection or at an appropriate time-point for any particular experimental setting.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Sterilization and post-hatch care of gnotobiotic Periplaneta americana, as adapted from Doll et. al. (see below)

Doll J, Trexler P, Reynolds L, Bernard G. 1963. The use of peracetic acid to obtain germfree invertebrate eggs for gnotobiotic studies. Am Midl Nat 69:231-239

[STEPS]
SECTION: Collect oothecae egg cases) from colony
?. 
SECTION: Physically clean any visible/major excrement off of the outside of the oothecae
?. 
SECTION: Wash oothecae in 0.1% SDS
?. 
SECTION: Rinse oothecae
?. 
SECTION: Prepare 0.1% peracetic acid for sterilization
?. 
SECTION: Sterilize oothecae
?. 
SECTION: Rinse oothecae (post-sterilization)
?. 
SECTION: Incubate eggs @ 30 C until hatching
?. 
SECTION: Post-hatch care
?.
TITLE: Production of magnetic GFP affinity beads using a Camelid anti-GFP nanobody
AUTHORS: Nichanok Auevechanichkul, Ruth Lintermann, Lennart Wirthmueller

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Our protocol for production of 'home-made' magnetic GFP affinity beads  using a Camelid anti-GFP nanobody. These can be used to immuno-purify GFP-  or YFP-tagged proteins from cell extracts.</div></div>

[STEPS]
TITLE: Lysogeny Broth (1L)
AUTHORS: Brian Smith, baltrus@email.arizona.edu micro.tucson

[STEPS]
?. [Fill container aproximately 750mL of H2O]
Add stir bar and set to mix
?. [Add ingredients]
[Bacto Tryptone]
[Bacto Yeast Extract]
[NaCl]
?. [Bring volume to 1L by adding H2O]
Allow to mix again with stir bar.
?. [Autclave]
Add agar to container to be autclavedThen add media
15 g
?. [Autclave]
Autclave at 121ºC for 30min
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The printed instructions accompanying each Foldscope kit are very detailed and easy to follow. Even easier is <a href="https://www.youtube.com/watch?v=wJtJJixMncA" target="_blank">this</a> excellent 14-minute video tutorial for the assembly. </p>
<p> </p>
<p>I didn't know about the video when I was assembling the Foldscope, and despite carefully following the printed instructions, I still managed to make a few embarrassing mistakes. I am sharing the protocol below with the comments on these mistakes, so others can avoid them. Hopefully, more new users will comment, pinpointing the assembly steps that turned out to be error-prone for them.</p>

[BEFORE_START]
<p>Do watch the <a href="https://www.youtube.com/watch?v=wJtJJixMncA" target="_blank">14-minute video</a> tutorial on assembling the Foldscope.</p>

[GUIDELINES]
<p> </p>
<p><strong>Components:</strong></p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/en5cz36.png" data-src="https://s3.amazonaws.com/pr-journal/en4cz36.png" data-ofn="Screen Shot 2016-08-11 at 3.27.11 PM.png" /></p>
<p><strong>S1 </strong>= main stage</p>
<p><strong>S2 </strong>= sliding stage</p>
<p><strong>P1 </strong>= LED holder</p>
<p><strong>P2 </strong>= paper slides</p>
<p><strong>P3 </strong>= magnet holder</p>
<p><strong>L1 </strong>= low magnification lense (small apertrure, large lense)</p>
<p><strong>L2 </strong>= high magnification lense (<strong>very</strong> small aperture, small lense)</p>
<p><strong>M1 </strong>= magnet strip for Foldscope (black adhesive tape)</p>
<p><strong>E1 </strong>= light module</p>

[STEPS]
?. 
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TITLE: DNA extraction (BOMB)
AUTHORS: Tsu-Chun  Hung, Yin-Tse    Huang

[DESCRIPTION]
DNA extraction (BOMB)

[STEPS]
SECTION: Sample Collection
1. Add 200 µL of 1mm beads to 2ml enppendorf tube

SECTION: Sample Collection
2. Add200 µL of 0.5mm beads to 2ml enppendorf tube

SECTION: Sample Collection
3. Add 225 µL of TE buffer to 2ml enppendorf tube
 

SECTION: Sample Collection
4. Add 375 µL of lysis buffer to 2ml enppendorf tube
 


SECTION: Sample Collection
5. Add 267 µL of 10M ammonium acetate to 2ml enppendorf tube
 

SECTION: Sample Collection
6. Collect 10-20 mg of sample to 2ml enppendorf tube
SECTION: Sample crush
7. Put 2ml eppendorf tube in mixmill for sample crush, at this condition: 30 rpm/s, for 4mins 4 min 
SECTION: Centrifugation
8. Put 2ml eppendorf tube in centrifuge for centrifugation, at this condition:17.0 x g, 3 min, 25 °C 
SECTION: DNA purification
9. Add 350 µL of isopropanol to the 1st well of 96 well plate

   

SECTION: DNA purification
9.1. Add 125 µL of magnetic beads (10 mg/ml) to the 1st well of 96 deep well plate 

 


SECTION: DNA purification
9.2. Add 200-300 µL of the sample (lysate) from the 2ml centrifuged tube to the 1st well of 96 deep well plate 
 

SECTION: DNA purification
10. Add 400 µL of isopropanol to the 2nd well of 96 deep well plate 
SECTION: DNA purification
11. Add 300 µL of 80% enthanol to the  3rd well of 96 deep well plate 
SECTION: DNA purification
12. Add 300 µL of 80% enthanol to the 4th well of 96 deep well plate 
SECTION: DNA purification
13. Add 100 µL of DD water to the 5th well of 96 deep well plate 
SECTION: DNA purification
14. Put the prepared 96 deep well plate in the automated DNA extraction machine

SECTION: DNA purification
15. After the extraction is done, collect 50 µL of the eluted sample as the DNA template for downstream experiments