eamag/protocols-with-steps huggingface dataset:
TITLE: Genomic DNA isolation from fixed cells
AUTHORS: Herschel Dhekne, Ebsy Jaimon, Suzanne R Pfeffer

[DESCRIPTION]
This protocol details the procedure of genomic DNA isolation from fixed cells.

[STEPS]
SECTION: Genomic DNA isolation from fixed cells
1. Resuspend 1-3 X106 cells in 250 µL of PBS and transfer to a 2 mL tube  (this would represent one   dish of 3T3 cells).
SECTION: Genomic DNA isolation from fixed cells
2. Add 200 µg Proteinase K (from a 20 mg/mL stock) and 200 µg RNase A (from a 20 mg/mL stock).
SECTION: Genomic DNA isolation from fixed cells
3. Incubate cells at 37 °C for 30 min in a water bath.
SECTION: Genomic DNA isolation from fixed cells
4. Add 250 µL Qiagen AL lysis buffer per 250 µL of the protease and RNAase-containing cell suspension and mix thoroughly.
SECTION: Genomic DNA isolation from fixed cells
5. Place tubes in an incubator at 56 °C with shaking at 800 rpm  , capped.
 



SECTION: Genomic DNA isolation from fixed cells
6. Add 250 µL, 100% molecular biology grade ethanol; mix slowly using a slow vortex for 5 s.
SECTION: Genomic DNA isolation from fixed cells
7. With a razor blade, trim the tip of a 1 mL pipet tip to enlarge the opening.  Use this tip to pipet out DNA from the ethanol solution and apply it onto a silica spin DNA binding column (e.g. EconoSpin 1920-250).
SECTION: Genomic DNA isolation from fixed cells
8. Spin at 6000 x g for 1 min in a fixed angle tabletop microfuge; aspirate and discard flow-through.
SECTION: Genomic DNA isolation from fixed cells
9. Add 500 µL Qiagen buffer AW1 to the column, spin again at 6000 x g for 1 min, aspirate and discard flow-through.
SECTION: Genomic DNA isolation from fixed cells
10. Add 500 µL Qiagen buffer AW2, spin at 8000 x g for 1 min, aspirate and discard flow-through.
SECTION: Genomic DNA isolation from fixed cells
11. Spin once more using microfuge  to remove excess ethanol at 13000 x g for 1 min.
SECTION: Genomic DNA isolation from fixed cells
12. Transfer column into a new, 1.5 mL collection tube.
SECTION: Genomic DNA isolation from fixed cells
13. Elute with pre-warmed, 100 µL nuclease-free water or TE.
 

SECTION: Genomic DNA isolation from fixed cells
14. Incubate for 1 min, then spin at 13000 x g for 1 min.
SECTION: Genomic DNA isolation from fixed cells
15. Add another 50 µL nuclease free water to accomplish a second elution.
SECTION: Genomic DNA isolation from fixed cells
16. Incubate at Room temperature  for 1 min, then spin as before at 13000 x g for 1 min.
SECTION: Genomic DNA isolation from fixed cells
17. The two flow-through fractions contain the genomic DNA.
SECTION: Genomic DNA isolation from fixed cells
18. Perform Nanodrop and Qubit HS DNA estimation to calculate yield.
TITLE: Virtual Screening
AUTHORS: BOC  Sciences

[DESCRIPTION]
Virtual screening has been widely applied in early-stage drug discovery. As an alternative or complementary approach to high-throughput screening (HTS) assays with high cost and low hit rate, virtual screeningis an efficient computational method to identify drug candidates in silico from large chemical compound databases. Its usefulness has been verified by current applications that successfully retrieved hit and lead identifications against various disease targets.

[STEPS]
TITLE: Grinding Tissue with the Qiagen Tissuelyzer (Soybean, Cowpea, Tobacco)
AUTHORS: Steven Burgess, Lynn Doran

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Grinding tissue using the Qiagen Tissuelyzer for DNA extraction, RNA extraction, or Immunoblotting.</div><div class = "text-block"><span style = "font-weight:bold;">Note</span><span>: when using the TissueLyser II it is recommended to use 2 mL centrifuge tubes in conjunction with 4mm beads. 1.5mL tubes are narrow at the end and samples will not grind properly if you use the 4mm beads.</span></div></div>

[STEPS]
?. Pre-cool components for tissue lysis.
?. Pre-cool and sterilize 4mm SPEX stainless steel grinding beads in 100% ethanol in a beaker at   for
-80 °C
?. Pre-cool the empty TissueLyser inserts for LN2 for  by lowering the inserts into a full liquid nitrogen dewar with the extra-long tongs.
Wear lab coat, goggles, cryogen safe gloves, and closed toe shoes.
***WARNING: The equipment manufacturer, Qiagen, does not support this practice. Manufacturer recommends precooling inserts at  in a ultra low freezer or on ground dry ice.  Manufacturer does not guarantee robustness or safety of equipment below  . ***Liquid nitrogen cooling  ( ) maintains highest sample integrity for nucleic acid extraction and analysis but there is a risk of damaging equipment and it will void any manufacturer warranties on equipment.
?. Prepare sample tubes for tissue lysis
?. Remove sample tubes from  storage or liquid nitrogen, if grinding immediately after sampling. Place samples in crushed dry ice.
-80 °C
?. Using forceps cooled in LN2, add one sterilized, pre-cooled SPEX bead per sample tube.
?. Remove Tissuelyser II cassettes from liquid nitrogen using extra-long tongs. Insert tubes into pre-cooled TissueLyser II cassettes, ensuring a balanced number of samples between cassettes.
?. Grind tissue for   at 20 Hz
Warning: Do not exceed this frequency, higher frequencies increase the number of cases where the steel beads will break the lid of centrifuge tubes resulting in sample loss. If using the SPEX 2150 beads it is necessary to use 2 mL centrifuge tubes to ensure proper grinding, in 1.5 mL tubes the bead will not reach the tapered bottom leaving samples unground
?. Remove cassettes from TissueLyser II, keeping samples in them, and submerge them in LN2 using extra long tongs to prevent thawing.  When liquid nitrogen has stopped bubbling, cassettes are ready to reinstall on the TissueLyser II.
Hold the cassette containing samples closed with the extra long tongs in the liquid nitrogen until bubbling has stopped or sample tubes may be dislodged from the cassette by the bubbling.
?. Grind tissue for   at 20 Hz
This repeat is to ensure all tissue is correctly grounded. There will be odd instances where leaf tissue has not properly ground for reasons such as samples sticking to the edge of tubes, check for this on removal, it may be necessary to repeat again.
?. Remove tubes with forceps cooled in LN2. Return to crushed dry ice for further analysis.  Alternatively, samples can be stored at   for several months before processing further.
-80 °C
TITLE: Electrocompetent Agrobacterium preparation
AUTHORS: Magdalena Julkowska

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Preparing electrocompetent Agrobacterium - used for the GV301 strain that we currently have in stock in Salt Lab</div></div>

[STEPS]
?. Grow seed culture to saturation (36-48 hours), you will need ~5mL
?. Inoculate 1 L of YEP media (20g peptone, 10 g yeast/ 1L) 1:200 with saturated agro. Alternatively inoculate 500ml YEP 1:100. Grow culture to an OD600  of 1.5 (24 hours or so). If you dont have YEP, just use normal LB media. Proceed on ice.
?. Spin cells at 5000 rpm for 15 minutes, resuspend (wash) in 1 volume cold,sterile H2O
?. Spin cells at 5000 rpm for 15 minutes, resuspend (wash) in 0.5 volume cold,sterile H2O
?. Spin cells at 5000 rpm for 15 minutes, resuspend (wash) in 0.02 volume cold,sterile 10% (v/v) glycerol.
?. Spin cells at 6000 rpm for 15 minutes, resuspend (wash) in 0.001 volume cold,sterile 10% (v/v) glycerol.  Pipette the glycerol onto cells and gently stir until well mixed.  The cells will be viscous. Final volume will be 0.003X original culture vol.
?. Dispense cells in ~80μl aliquots into screw top tubes—sample is viscous and challenging to pipette accurately, using a P1000 makes pipetting quicker and less difficult.  Place tubes in freezer box and freeze quickly by adding liquid nitrogen.
?. Store at –80°C.  Thaw on ice before use.
TITLE: Mouse brain slice electrophysiology
AUTHORS: Shiyi Wang

[DESCRIPTION]
Mouse brain slice electrophysiology

[STEPS]
1. **Animal Handling**
1.1. - Anesthetize 3-4 mice of each genotype (WT and LRRK2 G2019Ski/ki) and condition using 200 mg/kg tribromoethanol (Avertin).
1.2. - Ensure proper anesthesia depth and confirm absence of reflexes before proceeding.
2. **Brain Extraction and Slicing**
2.1. - Decapitate the mice and immediately immerse the brains in ice-cold artificial cerebrospinal fluid (aCSF) containing (in mM): 125 NaCl, 2.5 KCl, 3 mM MgCl2, 0.1 mM CaCl2, 10 glucose, 25 NaHCO3, 1.25 NaHPO4, 0.4 L-ascorbic acid, and 2 Na-pyruvate, pH 7.3-7.4 (310 mOsmol).
2.2. - Use a vibrating tissue slicer (Leica VT1200) to obtain 350 μm thick coronal brain slices containing the anterior cingulate cortex (ACC).
3. **Slice Incubation**
3.1. - Transfer slices immediately to standard aCSF maintained at 33°C and continuously bubbled with 95% O2 – 5% CO2.
3.2. - Incubate slices for 30 minutes at 33°C to recover.
4. **Transfer to Holding Chamber**
4.1. - After incubation, transfer slices to a holding chamber at room temperature (approximately 25°C) with the same extracellular buffer.
5. **Microscopy Setup**
5.1. - Visualize brain slices using an upright microscope (BX61WI, Olympus) equipped with a 40x water-immersion objective and infrared-differential interference contrast optics.
5.2. - Use a digital camera (ODA-IR2000WCTRL) for image acquisition.
6. **Patch-Clamp Setup**
6.1. - Perform patch-clamp recordings using an EPC 10 patch-clamp amplifier controlled by Patchmaster Software (HEKA).
6.2. - Set data acquisition at a sampling rate of 50 kHz and low-pass filter at 6 kHz.
7. **Recording Configuration**
7.1. - Use aCSF bath solution containing: For mEPSCs: 1 µM tetrodotoxin and 50 µM Picrotoxin, held at -70 mV in voltage-clamp mode; For mIPSCs: 1 µM tetrodotoxin, 10 µM CNQX, and 50 µM D-AP5, held at -70 mV in voltage-clamp mode.
8. **Internal Solution Preparation**
8.1. - Prepare internal solutions for patch pipettes:  For mEPSCs: 125 K-gluconate, 10 NaCl, 10 HEPES, 0.2 EGTA, 4.5 MgATP, 0.3 NaGTP, and 10 Na-phosphocreatine, pH adjusted to 7.2 – 7.4 with KOH, osmolality ~300 mOsmol. For mIPSCs: 77 K-gluconate, 77 KCl, 10 HEPES, 0.2 EGTA, 4.5 MgATP, 0.3 NaGTP, and 10 Na-phosphocreatine, pH adjusted to 7.2 – 7.4 with KOH, osmolality ~300 mOsmol.
9. **Data Acquisition and Analysis**
9.1. - Record mEPSCs and mIPSCs using Minhee Analysis software.
9.2. - Analyze frequency by counting events over 5 minutes of recording.
9.3. - Calculate average events per cell based on at least 100 non-overlapping events.
9.4. - Measure peak amplitude of average mEPSCs relative to baseline current.
TITLE: Feline Respiratory Pathogen Detection Assays
AUTHORS: Come J Thieulent, Mariano Carossino, Laura Peak, Udeni B. R. Balasuriya

[DESCRIPTION]
The Feline Respiratory Pathogen (FRP) Detection Assays is intended as an in vitro veterinary reagent set, based on quantitative PCR (qPCR) and Reverse Transcription qPCR (RT-qPCR), for the detection of feline calicivirus (FCV), feline herpesvirus type 1 (FHV-1), influenza A virus (IAV), SARS-CoV-2, Bordetella bronchiseptica, Mycoplasma felis and Chlamydia felis in nasal and pharyngeal swab samples.

[GUIDELINES]
SHIPPING & STORAGE INFORMATION
The FRP Detection Assays are shipped on dry ice. Reagents should arrive frozen. The Reagents in the purple and red tubes may arrive liquid, this will not result in a reduction in performance.

All reagents should be stored at -20°C upon arrival. All reagents can be stored for a minimum of one year (from the date of shipment) at -20°C without showing a reduction in performance. Positive controls should be stored at  -80°C.

LIMITATIONS:
Strict compliance with the instructions is required for optimal results.
Appropriate specimen collection, transport, storage, and processing procedures are required for the optimal performance of this test.
The presence of RT-PCR inhibitors may cause false negatives.
Results of FRP Detection Reagents need to be interpreted in consideration of all clinical and laboratory findings.

QUALITY CONTROL
The specificity of each test was validated using a panel of reference and related canine respiratory pathogens.
The analytical sensitivity of each assay was determined using ten-fold dilution of in vitro transcribed RNA or plasmid copies number. All assays have a limit of detection (LOD95)  15 copies/l.

[STEPS]
1. Thaw all reagents on ice.
2. Centrifuge all reagents on a benchtop centrifuge to ensure no liquid is in cap and keep on ice 
 

3. Setup the Master Mix according to the following table 1:
   
  

SECTION: PROGRAMMING THE THERMOCYCLER
4. Select the following fluorescence channels: ABYTM, Cy5, FAMTM, and VICTM.

 

SECTION: PROGRAMMING THE THERMOCYCLER
5. The standard mode should be selected, following the table below:
    

SECTION: RESULTS INTERPRETATION
6. Before analysis of results, the threshold value of each fluorescent dye must be manually set in the region of exponential amplification, typically 0.1 × ΔRn value at the plateau phase.
SECTION: RESULTS INTERPRETATION
7. Each assay is considered valid if the following criteria are met:
 
 
SECTION: RESULTS INTERPRETATION
8. The results are qualitative (Positive or Negative). A specimen is considered positive if the Ct value obtained is below the following Ct cut-off values:
TITLE: Lee et al. Digestion and Seeding (adapted from. Annika)
AUTHORS: Annika Fendler

[DESCRIPTION]
This Protocol is adapted from Lee et a. (2018) (https://www.sciencedirect.com/science/article/pii/S0092867418302976?via%3Dihub)

The authors have established organoids from clusters of bladder cancer cells after TURB. They use a medium with CS-FCS and EGF and culture the organoids as a sandwich in matrigel, The effiency was around 70%, genomics were comparable to the parental tumour but phenotypically the organoids tended to evolve into basal cells even when arising from a tumour with a luminal phenotype.


[STEPS]
SECTION: Prepare medium and enzyme mixes
1. Heat the shaking incubator to 37°C
SECTION: Prepare medium and enzyme mixes
2. Prepare and adequate volume of Lee Organoid Medium and HBSS Washing Solution.
SECTION: Thaw fragments
6. 
 

SECTION: Thaw fragments
7. Thaw 1 or more aliquots of fragments at 37 °C until only a small piece of ice remains

SECTION: Prepare medium and enzyme mixes
3. Thaw    o/n

SECTION: Prepare medium and enzyme mixes
4. Coat an adequate number of wells of a 24-well or 6-well plate with 60%   (diluted in organoid medium).

Add 75 µl or 250 µl of 60% Matrigel to each well. Keep the plate on ice and try to spread the   evenly with a pipette tip. Solidify for 15 min at 37°C

SECTION: Thaw fragments
8. Transfer fragments to a 50 ml Falcon and spin down 400 x g, 5 min . Remove the supernatant

SECTION: Thaw fragments
9. Transfer fragments to a glass petri dish and mince with a scalpell as fine as possible.
SECTION: Digestion of fragments
10. Transfer fragments to 1 mL to 5 mL of   
diluted 1:10 in Lee Organoid Medium.
See table 1 for the volumes used for each sample.

SECTION: Digestion of fragments
11. Incubate 15 min under gentle shaking at 37 °C .

SECTION: Prepare medium and enzyme mixes
5. Preaheat Lee Organoid Medium to 37°C
SECTION: Digestion of fragments
12. Spin down 350 x g, 5 min and discard supernatant.

SECTION: Digestion of fragments
13. Add 5 mL  of PBS and spin down 350 x g, 5 min  and discard supernatant.

SECTION: Digestion of fragments
14. Add 2 mL  to 5 mL  of    and incubate 3 min  at Room temperature 

SECTION: Digestion of fragments
15. Stop reaction with 5 mL HBSS Washing Solution.

SECTION: Digestion of fragments
16. Spin down for 350 x g, 5 min  and discard supernatant.

SECTION: Digestion of fragments
17. Resuspend in 2 mL of HBSS Washing Solution and pass through a   cell strainer.
Wash with 5 ml of HBSS Washing solution.

SECTION: Seed cell clusters as organoids
18. Spin down for 350 x g, 5 min  and carefully discard as much supernatant as possible.
SECTION: Seed cell clusters as organoids
19. Resuspend the cells in 75 µL or 250 µL  of 60%    and carefully layer over the matrigel coating of the prepared wells. Solidify for 15 min  at 37 °C .

SECTION: Seed cell clusters as organoids
20. Add 250 µL or 1.5 mL of Lee Organoid Medium per well.

SECTION: Results
21. Add text and pictures here to describe how organoids are growing. Please also reference the next protocol that you are using to continue culturing
TITLE: Separating the Protein of Interest from MBP after TEV Protease Cleavage (NEB #E8201)
AUTHORS: New England  Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;"><span>Both the MBP-tag and TEV Protease are polyhistidine-tagged for easy removal from the TEV Protease reaction. Loading the digest onto NEBExpress Ni Resin (</span><a href="https://www.neb.com/products/s1428--nebexpress-ni-resin" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">NEB #S1428</span></a><span>) sequesters both the MBP-tag and TEV Protease, thereby isolating the protein of interest in the column flow through.

</span></li><li style = "counter-reset:ol0;list-style-type:disc;">The binding capacity of NEBExpress Ni Resin is ≥ 10 mg/ml. The binding capacity will vary depending on the target protein, binding conditions and the accessibility of the His-tag.

</li><li style = "counter-reset:ol0;list-style-type:disc;">It is recommended to estimate the amount of the His-tagged MBP released by TEV Protease cleavage by first analyzing a sample by SDS-PAGE and comparing its band size and intensity to that of the provided MBP6 protein control.</li></ul></div></div>

[STEPS]
?. [Resin Preparation]
Gently shake the Ni Resin bottle to completely resuspend the slurry and transfer desired amount to a closed column; allow resin to settle.
?. [Resin Preparation]
Open column and allow storage buffer to flow through.
?. [Resin Preparation]
Equilibrate the resin by adding 5 column volumes (CV) of Binding buffer.
?. [Binding]
Load the TEV Protease digestion directly onto the equilibrated column and allow it to flow through the resin slowly (flow rate
?. [Binding]
Collect the flow through containing the protein of interest in a sterile tube.
?. [Binding]
Wash the column with 1 CV of Binding Buffer to maximize the yield of the target protein; collect the flow through.
?. [Binding]
Dialyze or buffer exchange target protein into desired storage buffer.
?. [Resin Cleaning]
Add  to the column and allow a contact time of  to .
[NaOH]
?. [Resin Cleaning]
Add  to the column and allow a contact time of  –  followed by 10 CV of water.
[NaCl]
?. [Storage]
For long-term storage, NEBExpress Ni Resin should be stored in 20% ethanol at 2 – 8°C.
TITLE: UC Davis - RadioImmuno Assay (RIA) Protocol
AUTHORS: Peter Havel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">The RIA (RadioImmuno Assay) is an assay method used for the quantification of various proteins. A standardized concentration of antibody specific to the analyte of interest is pipetted into test tubes. A standardized concentration of the analyte of interest that is labeled with a radioisotope (usually 125I) is added to the tubes. Standards and samples are pipetted into the tubes and the tubes are incubated. During the incubation period the standardized concentration of labeled analyte and the unknown concentration of analyte in the samples will compete for binding sites on the antibody. After the incubation period a precipitating reagent is added to the tubes and the bound antibodies are precipitated out using the double antibody-polyethyleneglycol precipitation technique. The tubes are centrifuged and the supernatant is aspirated and the precipitate is counted (using a gamma counter if 125I is used). If there is a low concentration of the analyte of interest in the samples, the labeled analyte will have a higher probability for binding to the antibody and thus there will be a higher count in the precipitate. If there is a high concentration of the analyte of interest in the samples there will be a lower count of labeled analyte in the precipitate. The counts and known concentrations of the standards are used to generate a standard curve, and the counts of the samples are used to interpolate quantitative concentrations for the analyte of interest from the standard curve.</div></div>

[STEPS]
?. Follow kit instructions for preparing standards and all reagents.
?. Follow kit instructions for volumes and incubation times. Pipet standards and samples . Pipet antibody and tracer. Incubate.
?. Follow kit instructions for volumes and incubation times. Add precipitating reagent. Incubate.
?. Centrifuge.
?. Aspirate supernatant.
?. Count precipitate with gamma counter.
TITLE: Immunofluorescence for pSer129 alpha-synuclein and total alpha-synuclein in mouse brain 
AUTHORS: Laura Volpicelli-Daley, mamenard

[DESCRIPTION]
This protocol details the immunofluorescence of brain section for phopho-serine129-alpha-synuclein and total alpha-synuclein

[STEPS]
SECTION: Day 1
1. Pre-warm sodium citrate buffer to 37 °C.

SECTION: Day 1
2. Place sections in netwell inserts (Fisher Scientific 07-200-213) in 6-well plate.
SECTION: Day 1
3. Wash 3X in cold 1X TBS for 5 min each with shaking (around 2 mL per well).
SECTION: Day 1
4. Incubate at 37 °C for 60 min in pre-heated sodium citrate buffer (about 2 mL/well).

SECTION: Day 1
6. Block for 60 min at 4 °C on shaker. Two mL of blocking buffer per well. Make sure sections are not folded on themselves.

 

SECTION: Day 1
7. Place in primary antibody solution 60 min at 4 °C, shaking. For sections that cover an entire mouse forebbrain with 40 3190 sections spaced 240 3190 apart we use 2 mL per well. If you are just using a couple sections, you can use 0.5 mL per well in a new 24 well plate. Make sure sections are not folded on themselves.


 

SECTION: Day 1
3.1. Wash in cold 1X TBS for 5 min (1/3).

SECTION: Day 1
3.2. Wash in cold 1X TBS for 5 min (2/3).
SECTION: Day 1
3.3. Wash in cold 1X TBS for 5 min (3/3).
SECTION: Day 1
5. Wash 3X in 1X TBS for 5 min each.
SECTION: Day 1
5.1. Wash in 1X TBS for 5 min(1/3).

SECTION: Day 1
5.2. Wash in 1X TBS for 5 min(2/3).

SECTION: Day 1
5.3. Wash in 1X TBS for 5 min(3/3).

SECTION: Day 2
8. Wash 3X in cold 1X TBS for 5 min each with shaking.
SECTION: Day 2
8.1. Wash in cold 1X TBS for 5 min with shaking (1/3).
SECTION: Day 2
8.2. Wash in cold 1X TBS for 5 min with shaking (2/3).
SECTION: Day 2
8.3. Wash in cold 1X TBS for 5 min with shaking (3/3).
SECTION: Day 2
9. Place in secondary antibodies for 120 min at 4 °C, shaking. Cover plate with foil to protect from light.


 

SECTION: Day 2
10. Wash 3X in 1X cold TBS for 5min each with shaking.
SECTION: Day 2
10.1. Wash in cold 1X TBS for 5 min with shaking (1/3).
SECTION: Day 2
10.2. Wash in cold 1X TBS for 5 min with shaking (2/3).
SECTION: Day 2
10.3. Wash in cold 1X TBS for 5 min with shaking (3/3).
SECTION: Day 2
11. Gently mount onto Superfrost plus slides in 1X TBS with a couple drops of 10% Tx-100.

 

SECTION: Day 2
12. Allow to dry, coverslip using ProlongGold.
TITLE: Preparing Cholesterol for use in NGM
AUTHORS: Bonnie Evans

[DESCRIPTION]
C. elegans requires cholesterol to progress through the four larval stages. 

[STEPS]
1. Prepare 5 mg/ml in Ethanol by resuspending the whole 250mg vial in 50 ml ~100% ethanol. 
2.  Filter sterilise using vacuum pump. 
3. Store at 4 degrees protected from the light