eamag/protocols-with-steps huggingface dataset:
TITLE: Live-cell imaging; cell death assay
AUTHORS: Minee-Liane Choi

[DESCRIPTION]
This protocol contains instructions for examining cell death in live-cell imaging.  

[STEPS]
1. Cell death is detected using Propidium iodide (PI, Thermo Fisher Scientific) or SYTOX™ Green (SYTOX, Thermo Fisher Scientific) which is excluded from viable cells but exhibits red (PI) and green (SYTOX) fluorescence following a loss of membrane integrity and Hoechst 33342 (Hoechst, Thermo Fisher Scientific). 
2. 20 μM PI or 500 nM SYTOX and 10 uM Hoechst are directly added to the dishes. 
3. Cells are incubated for 15 min. 
4. Live-cell imaging is performed using a confocal microscope.
 

5. Hoechst and PI are excited by 405 nm and 543 nm laser lines with the emission between 405 nm to 470 nm and 570 nm to 640nm respectively. 
6. SYTOX is excited by a 488 nm laser with emissions between 488 nm and 516 nm.
TITLE: Overall protocol for top-down LC-MS/MS of human lung tissue
AUTHORS: Jeannie  Camarillo, Bryon  Drown, Neil  Kelleher

[DESCRIPTION]
Overview description of the process for acquiring top-down LC-MS/MS data on human lung tissue

[STEPS]
SECTION: Tissue Collection
1. Lung tissue was provided by University of Rochester Medical Center. Tissue was collected and prepared according to the following: 

 

SECTION: Sample Preparation
2. Perform LC based Proteomics on adjacent tissue sections:

 

SECTION: Data Acquisition
3. 
 

SECTION: Sample Preparation
4.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
MinElute columns can also be used on any vacuum manifold with luer connectors (e.g., QIAvac 24 Plus or QIAvac 6S with Luer Adapters). The following protocol is designed to extract and purify DNA of 70 bp to 4 kb from standard or low-melt agarose gels in TAE or TBE buffer using vacuum-driven processing resulting in high end-concentrations of DNA. Up to 400 mg agarose can be processed per MinElute column.

[BEFORE_START]
The yellow color of Buffer QG indicates a pH &le;7.5. <br /><br />Add ethanol (96&ndash;100%) to Buffer PE before use (see bottle label for volume).<br /><br />Isopropanol (100%) and a heating block or water bath at 50&deg;C are required. <br /><br />All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a conventional table-top microcentrifuge.

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Streptavidin-CBD purification
AUTHORS: Manuela de las Casas

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The aim of this protocol is to obtain the purified streptavidin with cellulose binding domains on both sides from the transformed bacteria </span><span style = "font-style:italic;">E. coli</span><span>. Later on, this protein will be attached to a membrane as part of the detection system. The obtention of the proteins starts at this point with transformed bacteria (</span><span style = "font-style:italic;">E. coli</span><span>).</span></div><div class = "text-block">The previous steps to obtain the transformed bacteria and further steps to use the purified protein do not correspond to this protocol.</div></div>

[STEPS]
?. [Growing the cells]
From the Petri dishes with the bacteria, pick a few colonies with a Digralsky planting handle and resuspend the colonies in liquid LB in an Erlenmeyer flask. It is recommended to set more than one flask, to minimize risks in case one of the flasks goes wrong. Grow a negative control as well (the bacteria without the plasmid).
?. [Growing the cells]
Let the bacteria grow in an incubator at 37ºC until it reaches an OD600 of 0.6 approximately (range 0.4-0.8). Check periodically the OD with the ultrospec. To check the OD, set a blank cuvette with just LB to compare the bacteria grow. Be careful not to overgrow the bacteria. If overgrown, they won't be useful for transformation. The culture should be at log phase.The volume pipetted into the cuvettes is discarded after the measurements. Do not return it to the flask.
?. [Induction of the expression]
Since the promoter used for this bacteria has a constitutive expression, there is no need to induce. But depending on the promoter you used, you might need to induce the expression of the protein.In our case, we just need to let the bacteria grow as step 1.1 indicates.
?. [Purification of the protein]
To purifiy the protein, it is necessary to lysate the cells. In order to do so:
?. [Purification of the protein]
Pick 1-2 mL of bacteria, centrifugate 3-5 min at 15000 x g. After centrifugation, take out the supernatant carefully and resuspend the cells pellet with B-mercaptoethanol and SDS.
?. [Comprobation of the procedure]
OPTIONAL. This step is not necessary to obtain the protein. But with the purified protein, it is recommended to check that the procedure has gone right and that we have the desired protein.Boil the samples 3-5 min at 98ºC-100ºC. After boiling, centrifugate again for 3-5 min at 15000 x g. Run an electrophoresis (follow an electrophoresis protocol). Recommended to use 1:10 and 1:100 dilutions from the supernatant in an SDS-page acrylamide gel (pore size 10-12%). Materials and steps for the electrophoresis are not contemplated in this protocol.
TITLE: Sequential smFISH
AUTHORS: ZengU19 BICCN Grant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">We have developed a multiplexed single molecule FISH protocol for use at the Institute. This protocol was optimized on human tissue, but will work on mouse tissue as well. It was adapted from Lyubimova et. al., Nature Protocols, 2013. </div></div>

[STEPS]
?. [Tissue and Sectioning ]
10-14 um cryosections are taken from fresh-frozen tissue, which are collected on poly-lysinetreated #1 coverslips at room temperature (RT). After 5-10 min at RT, sections are placed at 4°C until sectioning is complete. At that point, proceed immediately to fixation and permeabilization.
[RT]
?. [Fixation/Permeabilization]
Post-fix sections for 15 min with 4% PFA @ 4 °C.
[Post-fixing]
[Post-fixing]
?. [Fixation/Permeabilization]
Wash with PBS (1/3)
?. [Fixation/Permeabilization]
Wash with PBS (2/3)
?. [Fixation/Permeabilization]
Wash with PBS (3/3)
?. [Fixation/Permeabilization]
Permeabilize with cold methanol at -20 C for 10 min.
[Permeabilizing]
[Permeabilizing]
?. [Fixation/Permeabilization]
Air dry for 30 min in fume hood (Stopping point: store coverslips at -80C)
[Air drying]
?. [Fixation/Permeabilization]
Optional: Treat sections with 8% SDS/PBS for 10 minutes, followed by 3 – 5 rinses with PBSor 2XSSC
?. [Fixation/Permeabilization]
Add 2ml 2X SSC
[2X SSC]
?. [Hybridization]
Pre-heat hyb oven to 37 °C
[oven]
?. [Hybridization]
Place sections in hyb buffer without probes.
?. [Hybridization]
Add 4 ul probe 400ul hyb buffer.
[probe]
[hyb buffer]
Specific to 6-well plate format – if using perfusion chamber, this volume can be reduced.
?. [Hybridization]
Incubate at 37 C for 2H.
[Incubation]
[Incubation]
?. [Wash]
Add 2 ml wash buffer to each well.
[wash buffer]
?. [Wash]
Incubate at 37 C for 15 min.
[Incubation]
[Incubation]
?. [Wash]
Remove wash buffer.
?. [Wash]
Add 2 ml fresh wash buffer and incubate at 37 C for 15 min.
[wash buffer]
[Incubation]
[Incubation]
?. [Wash]
Replace wash buffer with fresh wash buffer + DAPI (final 5ug/mL) and incubate at 37 C for 15 min.
[Incubation]
[Incubation]
?. [Wash]
GLOX buffer step if performing antibody stain
?. [Wash]
Mount and image or store at 4 C in 2XSSC until imaging session
?. [Stripping]
65% formamide/2X SSC, 10 min X 3, 30 C
?. [Stripping]
Wash in 2XSSC (1/3)
?. [Stripping]
Wash in 2XSSC (2/3)
?. [Stripping]
Wash in 2XSSC (3/3)
?. [Stripping]
Following stripping, proceed to hybridization step.
[Hybridization step]
TITLE: Canary VOC testing protocol
AUTHORS: Charlie Mize, Raj Reddy, Purav Badani

[STEPS]
?. 
?. Turn on Basestation.  If Basestation is already on because a test has just been completed, leave the Basestation on.  Attach disposable sensor cartridge (or Sensor Tray Housing, STH) to Basestation.  Device wil run self-test.
?. Testing subject breathes into the breathing tube of the STH at a normal minute ventilation for 1-3 minutes.
Staff testing patient should wear PPE to prevent infection.  Patient will exhale into device for both testing and safety.  The sensor cartridge will be a closed system designed to guide exhaled breath towards the outlet.  All air outlets incorporate HEPA filters to prevent inadvertent escape of virus.
?. Testing result will be read on the device as POSITIVE, NEGATIVE or INCONCLUSIVE/ERROR.
?. Report result to testing subject.  Remove and dispose of STH.  Begin new test, beginning with No. 1 above.
TITLE: Scratch-wound assay
AUTHORS: Goran  Tomic

[DESCRIPTION]
This protocol describes the process for setting up a scratch-wound assay analysed by the IncuCyte system.

[STEPS]
1. 
Seed cells at a density of 20,000 cells/well (might need optimisation for a particular cell line) in ImageLock 96-well plates. I usually plate replicates in columns as that way you can easily calculate the mean and export from the IncuCyte software directly.
2. Incubate overnight (12-16 hours) or until confluent (make sure you always keep the same incubation time for consistency)
3. Create the wound using the WoundMaker: 
Remove top of WoundMaker and set top in empty wash boat.    
Rinse the Wound Maker in 70% ethanol, PBS and then use for making the wound. Rinse with ethanol after use. Never flip it over.                          
Insert plate (containing cells & media) into base plate holder. Remove plate cover.                      
Replace pin block by guiding the rear dowels of pin block into the rear holes of the base plate. Do not push down.                   
Push and hold the black lever.                      
Lift pin block while continuing to hold the black lever down.                      
Replace plate cover. Wash wells (up to two washes). 
4. Immediately aspirate the media from each well and carefully wash the cells twice with culture media (100 µL/well). Be gentle while adding the medium and aspirating as the confluent layer of cells might easily detach).
5.  After washing, add 100 µl of culture media
6. Place the cell plate into the IncuCyte live-cell analysis system and allow the plate to warm to 37°C for 30 minutes prior to scanning. 
a. Objective: 4x, 10x (recommended), or 20x
b. Channel selection: Phase Contrast (+ Fluorescence if analyzing cells with fluorescent labels)
c. Scan type: Scratch Wound (Wide Mode optional for 10x, required for 20x)
7. Incubate overnight and check if wound is closed.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>BioLegend MojoSort™ nanobeads work in commonly used separation columns, based on our internal research as well as validation by external testing by academic labs. This simple protocol consists of following the MojoSort™ protocol to label the cells with pre-diluted MojoSort™ reagents and using the columns as indicated by the manufacturer.</p>
<p> </p>
<p>Note: Due to the properties of our beads, it may be possible to use far fewer beads that with other commercial suppliers. We recommend a titration to find the best dilution factor. However, as a general rule, dilutions ranging from 1:3 to 1:20 for the Nanobeads can be used.</p>

[GUIDELINES]
<p><strong>Commercially available columns:</strong></p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/dsiecje.jpg" data-src="https://s3.amazonaws.com/pr-journal/dshecje.jpg" data-ofn="mojosort positive selection.JPG" /></p>
<p> </p>
<p><strong>Example of magnetic separation with medium capacity columns:</strong></p>
<p><br />a) Place the column in a magnetic separator that fits the column.<br />b) Wash the column with 3 mL of buffer.<br />c) Add the labeled cell suspension to the column through a 30 μm filter and collect the fraction containingthe unlabeled cells.<br />d) Wash the column 3 times with 3 mL of buffer and collect the fraction containing the unlabeled cells.Combine with the collected fraction from step c. These cells may be useful as controls, to monitorpurity/yield, or other purposes.<br />e) Take away the column from the magnet and place it on a tube. Then add 5 mL of buffer and flush outthe magnetically labeled fraction with a plunger or supplied device. These are the positively isolatedcells of interest; do not discard. To increase the purity of the magnetically labeled fraction repeat theisolation process with a new, freshly prepared column.</p>
<p> </p>
<p><strong>View protocol on Biolegend.com for <a href="http://www.biolegend.com/media_assets/support_protocol/MojoSort_Positive_Selection_Columns_01052015.pdf" target="_blank">Data</a></strong></p>

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

[DESCRIPTION]
<p>Backgrounds and aims: The association of melanosis coli with the development of</p>
<p>colorectal polyps remains uncertain.</p>
<p> </p>
<p>Methods: From a total of 18263 patients who had received colonoscopy in our hospital,</p>
<p>219 with melanosis coli cases and 438 controls matched by age and sex (at 1:2 ratio)</p>
<p>were included in this study. The association of incidence, number, location, and</p>
<p>pathology of colorectal neoplasm with grades and distribution of melanosis coli were</p>
<p>analyzed.</p>
<p> </p>
<p>Results: Melanosis coli was associated with significantly more colorectal polyps than</p>
<p>control, a higher incidence of numerous colorectal polyps (number ≥ 20) (7.3% vs</p>
<p>0.5%; p &lt; 0.001), and higher number of small colorectal polyps (diameter ≤ 5 mm; p &lt;</p>
<p>0.01). Patients with melanosis coli had higher incidences of low-grade adenomas</p>
<p>(31.1% vs 23.3%, p &lt; 0.05) and non-adenoma polyps (20.1% vs 12.8%, p &lt; 0.05) than</p>
<p>the controls. On multivariate analysis, melanosis coli was independently associated</p>
<p>with increased detecting rates of low grade adenoma (OR = 1.54; 95%: 1.06-2.23; p &lt;</p>
<p>.05), non-adenoma polyp (OR = 1.72; 95%: 1.11-2.70; p &lt; .05) and numerous polyps</p>
<p>(OR = 16.2, 95%: 3.66-71.6; p &lt; .05). There was no significant difference in the</p>
<p>incidence of high-grade adenomas or adenocarcinomas in the two population groups,</p>
<p>but the numbers of these lesions were insufficient to permit firm conclusions. No</p>
<p>significant differences in incidence, number, and pathology of colorectal polyps</p>
<p>between individuals with melanosis coli of three different grades of severity were found.</p>
<p>Melanosis located predominantly in the right colon had an interestingly lower incidence</p>
<p>of colonic polyps in right colon than did melanosis located predominantly in the left</p>
<p>colon or total colon (8.9% vs. 26.3%, 24.0%, p &lt; 0.05). Patients with melanosis coli</p>
<p>had significantly more nonspecific distal ileal ulcers than did controls (8.0% vs 0%, p &lt;</p>
<p>0.001).</p>
<p> </p>
<p>Conclusion: Melanosis coli is associated with a higher incidence and number of colonic</p>
<p>non-adenoma polyps and low-grade adenomas, and higher incidence of distal ileal</p>
<p>ulcers. Melanosis coli may not be a harmless pigmentation, but a sign of chronic injury</p>
<p>of colonic and intestinal mucosa.</p>
<p> </p>

[STEPS]
?.
TITLE: Endometrium dissociation with collagenase
AUTHORS: Roser Vento-Tormo, Regina Hoo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describe the tissue dissociation procedures from human endometrium and pregnancy endometrium samples. This protocol is adapted from</span><span style = "font-style:italic;"> Vento-Tormo et al. 2018 Nature</span><span> with some modification from Prof. Ashley Moffett (Department of Pathology, University of Cambridge). </span></div></div>

[STEPS]
?. [Tissue dissociation and digestion]
Note: Flash-frozen tissue with isopentane for Spatial Transcriptomics work. {optional} Note: If tissue is going to be transported, do it with preservation solution (HypoThermosol® FRS) at  . Store sample for fixing in formalin (RNA Scope) & nuclei sequencing (flash-frozen) {optional}.Scrape off the blood vessels on remaining tissues. Note: We can skip this step if the donor is perfused.
4 °C
?. [Tissue dissociation and digestion]
Place wet tissue under a petri dish. Take 2 scalpels and roughly mince up the tissue. This step is crucial to increase the efficiency of the digestion.
?. [Tissue dissociation and digestion]
Transfer contents to 50ml falcon containing the collagenase mix (~  /tissue but it will depend on the size of the tissue)
3 ml
?. [Prepare collagenase mix]
Collagenase mix recipe:   ABCD1ProductStockFinal volume (20ml : 3ml/sample)Concentration2RPMI or Hams F12 + 10% FBS9 ml RPMI or Ham's F12 + 1 ml FBS8.9 ml3Collagenase V10 mg/ml1 ml1 mg/ml4DNase I10 mg/ml100 ul0.1 mg/ml
ABCD1ProductStockFinal volume (20ml : 3ml/sample)Concentration2RPMI or Hams F12 + 10% FBS9 ml RPMI or Ham's F12 + 1 ml FBS8.9 ml3Collagenase V10 mg/ml1 ml1 mg/ml4DNase I10 mg/ml100 ul0.1 mg/ml
?. [Tissue dissociation and digestion]
Tighten lid and then seal with parafilm.
?. [Tissue dissociation and digestion]
Incubate at  for  . Shacking during the incubation is recommended.
37 °C
?. [Tissue dissociation and digestion]
Resuspend with   RPMI 10%.
20 ml
?. [Tissue dissociation and digestion]
Filter sample through small strainer (100um) – do not discard retained tissue. The retained tissue will be used for "Endometrium-Trypsin" protocol.
?. [Tissue dissociation and digestion]
Filtered material: Centrifuge at 450 g,   (0.5 rcf,  ).
?. [Tissue dissociation and digestion]
Wash with  of PBS twice.
10 ml
?. [Tissue dissociation and digestion]
Resuspend with RPMI 10%, count cells and sort for cell population (refer to "Cell staining for flow cytometry and sorting" protocol)
1 ml
?. [Tissue dissociation and digestion]
{optional- for biopsies we do this} Resuspend sample with  to  of 1X RBC lysis buffer mix and incubate for . *RLB preparation: Dilute 10X RLB stock with water. After RBC lysis, add of RPMI 10% and centrifuge 450g for Wash twice with 10ml of PBS.
2 ml
4 ml
10 ml
?. [Tissue dissociation and digestion]
{Optional} Resuspend cells in freezing medium to a concentration of 1x 107  cells and aliquot into cryogenic storage vials.
?. [Tissue dissociation and digestion]
Wash tissue with PBS {optional}.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is to annotate the tetraploid coffee long read transcriptome. </p>

[GUIDELINES]
<p><strong>Warning</strong></p>
<p>BLAST output format should be type 5 (.xml).</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Preparing 10x PCR buffer
AUTHORS: Nadine   Mowoh, Stephane  Fadanka

[DESCRIPTION]
10X ThermoPol Reaction Buffer is optimized for use with Vent® and Deep Vent® DNA Polymerases. This buffer also provides superior reaction conditions for other thermophilic DNA polymerases, includingTaqDNA Polymerase, OpenVent as well as various other DNA and RNA modifying enzymes.

Here we describe the protocol used in making a 10x PCR buffer recipe that provides optimum reaction condition for OpenVent polymerase enzyme. 

10x PCR buffer comprise of:

100 mM KCL
100 mM (NH)2SO4
200 mM Tris-HCL pH8.8 @ 25c
20 mM MgSO4
1 % Triton x100

[BEFORE_START]
Make sure all chemicals are available in their right concentrations needed to make the 10x PCR buffer.

[GUIDELINES]
This protocol describes the steps in preparing a low cost 10x PCR buffer.

[STEPS]
SECTION: Preparing reagent stocks
1. Before preparing the 10x PCR buffer, prepare 1M and initial stocks of the different chemicals that would be used.

 

 


Prepare 10 mL of 1 M concentration of each of the chemical stocks by weighing the salt powders as indicated in the table 1 below:
Weigh out the salts as indicated in the table and add to a 100 mL glass beaker.
Measure out 10 mL of distilled water and add to the glass beaker.
Add a magnetic flea and place on a magnetic stirring plate to mix the solution as shown in figure 1
To sterilize, autoclave the solutions by using a steam autoclave at 121° C for at least 30 minutes under at least 15 psi. (All the solutions should be transferred into Duran bottles that can withstand heating).

 


To make 1M Tris HCL pH 8.8

Weigh out 1.211 g Tris and add to a 50 mL glass beaker.
Measure out 8 mL of distilled water and add to the glass beaker.
Add a magnetic flea and place on a magnetic stirring plate to mix the solution as shown in figure 1.
Add a pH meter into the solution to observe the pH.
Slowly add the 3 M hydrochloric acid (HCl) solution using a Pasteur pipette to reduce the pH to pH 8.8. Be careful not to add too much at a time, since the pH will change rapidly.
Once the desired pH has been reached, top up the solution to 10 mL using distilled water.
 



SECTION: Pipetting to make  10x PCR buffer - 1mL
2. Pipette the following reagent stocks into a clean 1.5 mL Eppendorf tube as described in the table below to compose 1 mL of 10x PCR buffer.
1M KCL
1M (NH)2SO4
1M Tris-HCL pH8.8 @ 25c
1M MgSO4
Triton x100

 
Cork the tube and invert several times to mix the solutions
Label the tube appropriately and store at -20c.
TITLE: Antibiotic Stocks
AUTHORS: Alba Balletbó

[STEPS]
?. Unless otherwise indicated, the antibiotic powder can be dissolved in dH20. Addgene recommends making 1000X stock solutions and storing aliquots at -20ºC.  ABC1AntibioticRecomended Stock ConcentrationRecomended Working Concentration2Ampicillin100 mg/mL100 µg/mL3Chloramphenicol25 mg/mL(dissolve in EtOH)25 µg/mL4Gentamycin10 mg/mL10 mg/mL5Kanamycin50 mg/mL50 µg/mL6Spectinomycin/Streptomycin50 mg/mL50 µg/mL7Tetracycline*10 mg/mL10 µg/mL 'Note': All are dissolved in MQ except Chloramphenicol, which is dissolved in ethanol. All antibiotic mixes were filter-sterilized before use.
ABC1AntibioticRecomended Stock ConcentrationRecomended Working Concentration2Ampicillin100 mg/mL100 µg/mL3Chloramphenicol25 mg/mL(dissolve in EtOH)25 µg/mL4Gentamycin10 mg/mL10 mg/mL5Kanamycin50 mg/mL50 µg/mL6Spectinomycin/Streptomycin50 mg/mL50 µg/mL7Tetracycline*10 mg/mL10 µg/mL
?. To use, dilute your antibiotic into your LB medium at 1:1,000. For example, to make 100 mL of LB/ampicillin growth media, add 100 μL of a 100 mg/mL ampicillin stock (1000X stock) to 100 mL of LB.
?. Weigh the antibiotic powders needed for 10 mL of Recommended Stock Concentration Solution.
?. Filter sterilize and aliquot into 1.5 mL tubes.
?. Add the weighed powder to the MQ or ethanol solution. Mix/vortex vigorously until the powder is completely dissolved.
TITLE: UC Davis - Alanine transaminase
AUTHORS: Peter Havel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">
Alanine transaminase (ALT) also known as alanine aminotransferase or (sGPT) is a metabolic enzyme expressed primarily in the liver. Elevation of ALT levels is an indication of liver damage and has been associated with liver injury. ALT levels are monitored routinely in patients with liver diseases. ALT is also a very useful tool for preclinical investigation of experimental drug formulations and ALT levels are commonly used to monitor and attenuate the hepatotoxic effects of experimental drugs in rodents.</div></div>

[STEPS]
?. Add 10 μl of each sample or standard (in duplicate) to the microplate wells.IMPORTANT: Make sure not to add any bubbles to the wells when dispensing reagents, this will interfere with reading in the platereader.
?. Add 50 μl of ALT Reagent Solution to the wells. Cover wells with the adhesive film and incubate at 37°C for 30 min.
?. Carefully remove adhesive film and add 50 μl DNPH Color Solution to the wells. Use second film to re-cover wells and incubate for 10 min at 37°C.
?. Remove adhesive and add 200 μl 0.5 M NaOH to each well. Read 510 nm absorbance in plate reader.
?. A standard curve can be constructed using the serially-diluted standards by plotting the average absorbance for each oxaloacetate standard against its concentration in U/l. Calculate unknowns from standard curve.