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running a polyacrylamide gel in australia

  • Running agarose and polyacrylamide gels

    2011-6-17 · Polyacrylamide gels are used to separate shorter nucleic acids, generally in the range of 1−1000 base pairs, based on the concentration used (Figure 1). These gels can be run with or without a denaturant. Gels that are run without a denaturant are referred to as native gels.

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  • Polyacrylamide Gel Electrophoresis | Cleaver Scientific

    2018-3-5 · Place gel in a staining tray with 100 ml of fixing solution (40% ethanol, 10% acetic acid). Cover the tray, place on a rocker, and agitate gently for at least 2 hr. Pour off the fix solution and add 50 ml of 1x stain solution (dilute 1 part Flamingo Fluorescent Gel Stain with 9 parts diH. 2O).

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  • A Guide to Polyacrylamide Gel Electrophoresis and Detection

    2019-2-5 · polyacrylamide around gel. 19. Hold two pieces of dry 46 × 57–cm blotting paper together as one piece. Beginning at one end of gel and working slowly towards the other, lay paper on top of gel. Take care to prevent air bubbles from forming between paper and gel. 20. Peel blotting paper up; gel should come off plate with it. Gradually curl paper and

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  • Denaturing Polyacrylamide Gel Electrophoresis

    2013-11-4 · acrylamide gel solution to fill the gel mold completely. Make sure that no acrylamide solution is leaking from the gel mold. 6. Allow the acrylamide to polymerize for 30-60 minutes at room temperature. 7. After polymerization is complete, surround the comb and the top of the gel with paper towels that have been soaked in 1x TBE. Then seal the

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  • DNA Polyacrylamide Gel Electrophoresis

    2018-8-22 · Heat the gel (buffer) during the whole run at 60-70°C. 9. Wash the wells with 1X TBE buffer to remove UREA and gel pieces. 10. Load the samples. 11. Run the gel at 6 V/cm till the lower dye front reaches the three thirds of the gel. 12. Soak the gel for about 15 min in 1X TBE to remove the urea prior to staining. 13.

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  • Denaturing Polyacrylamide/Urea Gel Electrophoresis

    Gels are used in a wide variety of techniques, including Northern blotting, primer extension, footprinting, and analyzing processing reactions. They are invaluable as preparative and fractionating tools. There are two common types of gel: polyacrylamide and agarose. For most applications, denaturing acrylamide gels are most appropriate.

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  • Polyacrylamide gel electrophoresis of RNA

    2018-4-20 · 4. Running Gels 1. Load about 1 µL of PCR product into each well of the polyacrylamide gel. Load a DNA size ladder to both sides of the gel along with samples of PCR products. Fix the safety cover to the upper buffer chamber. 2. Connect the leads to the power supply, matching the color-coded red to red and black to black.

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  • Markers using a Non-denaturing Polyacrylamide Gel A Fast ...

    2000-12-1 · Adenovirus type 2 was dissociated with SDS and run on a 3% polyacrylamide gel containing 0.25% SDS in the Davis and Ornstein, high-pH, discontinuous buffer system, and stained with Amido Black. (b) Adenovirus type II dissociated with SDS and β-mercaptoethanol, run on a 0.1 m phosphate-buffered, 5% polyacrylamide gel containing 1% SDS, and stained with Coomassie …

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  • SDS polyacrylamide gel electrophoresis: Trends in ...

    Advantages of Near-Infrared In-Gel Westerns. Save time and reduce cost of running a blot; Eliminate variables associated with membrane transfer and blocking; NIR In-Gel Westerns provide sensitivity comparable to chemiluminescent In-Gel detection with clearer, sharper bands (Figure 2), but not as high as traditional NIR membrane Western detection.

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  • Overview of Electrophoresis | Thermo Fisher Scientific - AU

    Polyacrylamide gel electrophoresis in progress. Prepared gel cassettes are inserted into a gel tank, in this case the Invitrogen Mini Gel Tank, which holds two mini gels at a time.After wells are loaded with protein samples, the gels submerged in a conducting running buffer, and electrical current is applied, typically for 20 to 40 minutes.

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  • A Guide to Polyacrylamide Gel Electrophoresis and Detection

    2018-3-5 · Polyacrylamide Gel Electrophoresis (PAGE) When electrophoresis is performed in acrylamide or agarose gels, the gel serves as a size-selective sieve during separation. As proteins move through a gel in response to an electric field, the gel’s pore structure allows smaller proteins to travel more rapidly than larger proteins (Figure 2.1).

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  • Markers using a Non-denaturing Polyacrylamide Gel A Fast ...

    2018-4-20 · Pour appropriate amount (40 mL) of 6% non-denaturing polyacrylamide gel solution to a beaker for the plate set of 33 cm width × 10 cm height and spacer thickness of 1.5 mm, add 20 µL of tetramethylethylenediamine (TEMED) and 200 µLfresh 20% APS and mix gently.

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  • Bio 6 – SDS-PAGE Lab

    2017-2-3 · Part 1: Running an SDS-PAGE gel To begin this lab you will denature and load several different protein samples on a polyacrylamide gel after which you will run the gel as described. Preparing an acrylamide gel for SDS-PAGE is a bit tricky, so the polyacrylamide gels have been prepared for you. To denature your proteins it is essential that you

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  • SDS Polyacrylamide Gel Electrophoresis

    2011-7-14 · SDS Polyacrylamide Gel Electrophoresis Gel Recipes % Acrylamide 5% 7.5% 10.% 12.5% 15% 18% 4% Stacking Gel ... Makes ~30.8 ml gel solution for running gel; ~10 ml for stacking gel Electrophoresis Buffer: 5X Buffer: 1 X Buffer 60 g Tris base 9 g Tris base 288 g Glycine 43.2 g Glycine 50 ml 20% SDS 7.5 ml 20% SDS dH 2

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  • Purificationof DNA using nondenaturing polyacrylamide

    2016-8-9 · Shorterelectrophoresis times may be achieved by running the gel at higher voltagein a cold room so long as the temperature of the gel remains below thedenaturation temperature of the sample. Run the gel until the desired resolutionhas been obtained as determined empirically or from Table 2.7.1. ... Thepore size of a polyacrylamide gel is ...

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  • The principle and method of SDS-polyacrylamide gel ...

    Polymerized acrylamide (polyacrylamide) forms a mesh-like matrix suitable for the separation of proteins of typical size. The strength of the gel allows easy handling. Polyacrylamide gel electrophoresis of SDS-treated proteins allows researchers to separate proteins based on their length in an easy, inexpensive, and relatively accurate manner.

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  • SDS polyacrylamide gel electrophoresis: Trends in ...

    2000-12-1 · Fig. 1 SDS polyacrylamide gel electrophoresis of adenovirus type 2. (a) The first gel (7 July 1963) in which SDS was used. Adenovirus type 2 was dissociated with SDS and run on a 3% polyacrylamide gel containing 0.25% SDS in the Davis and Ornstein, high-pH, discontinuous buffer system, and stained with Amido Black.

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  • VERY SLOOOOOW running gels (SDS-PAGE) - Protein

    2007-11-14 · The problem is that the gels seem to run extremely slowly. I'm running a 12% gel right now (SDS-PAGE, 3.5% stacking, 12% sep) and I'm over 1 hour into the run with easily another hour to go, if not longer to go. The sample/dye has just barely made it into the separating gel. I'm running at 100 volts (anything higher and the buffer will be too hot).

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  • Purificationof DNA using nondenaturing polyacrylamide gel ...

    2016-8-9 · Shorterelectrophoresis times may be achieved by running the gel at higher voltagein a cold room so long as the temperature of the gel remains below thedenaturation temperature of the sample. Run the gel until the desired resolutionhas been obtained as determined empirically or from Table 2.7.1. ... Thepore size of a polyacrylamide gel is ...

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  • Assembling, loading, and running gels

    2012-11-18 · The assembly of a gel running stand varies with the type of apparatus. The top of the cassette must be continuous with an upper buffer chamber and the bottom must be continuous with a lower chamber so that current will run through the gel itself. ... A commonly used stain for detecting proteins in polyacrylamide gels is 0.1% Coomassie Blue dye ...

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  • The principle and method of polyacrylamide gel ...

    2018-7-20 · Agarose and Polyacrylamide Gels Dye Migration: Polyacrylamide Nondenaturing Gels. Dyes will migrate to the same point as double-stranded DNA of the indicated size in a nondenaturing polyacrylamide gel. Gel % Bromophenol Blue Xylene Cyanol 3.5 100bp 460bp 5.0 65bp 260bp 8.0 45bp 160bp 12.0 20bp 70bp 15.0 15bp 60bp 20.0 12bp 45bp

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  • Agarose and Polyacrylamide Gels - Promega

    Contact 1800 959 970 +61 8 6160 9073. Mon - Fri: 8am - 8pm EST Sat: 9am - 5pm EST Sun: Closed. [email protected]

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  • Gels & Caffeine Strips - Running Warehouse Australia

    2015-11-17 · Within a certain range determined by the porosity of the gel, the migration rate of a protein in the running gel is inversely proportional to the logarithm of its MW. Seen from the principle above, main advantages of discontinuous polyacrylamide gel electrophoresis is that when the protein samples go through the stacking gel, they can form a ...

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  • Principle and Protocol of Sodium Dodecyl Sulphate ...

    2008-5-23 · Stacking gel master mix: 340 ml H2O 62.5 ml 1.0 M Tris pH 6.8 5 ml 10% SDS Pouring resolving gel: 1. Make 6 ml of resolving gel (makes 1 gel, with a little bit leftover) 3.96 ml of resolving gel master mix 1.98 ml of 30% acrylamide 60 μl of 10% APS 2.4 μl of TEMED 2. Immediately load gel mixture into the casing with a pipette – fill to the ...

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  • 10% Acrylamide Gels for SDS-PAGE

    2018-3-2 · The stacking gel solution was prepared to 4% acrylamide, whereas the resolving gel contained 15% acrylamide, 9 M urea at pH 8.9. A Mini PROTEAN cell (Bio-Rad) filled with 0.02 M Tris, 0.19 M Glycine, pH 8.3 running buffer was used. Separation was performed at 4 °C, 0.01 A for 15 min, followed by 0.03 A for 2 h.

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  • Use of urea-polyacrylamide electrophoresis for ...

    2001-6-22 · 8. Fix gel. Cut gel down to sample lanes + 1-2 lanes each side. Fix gel in 3 X 5 minute washes of 10% MeOH, 10% AcCOOH (dilute 5X destain 1 in 5). Use ~10 X gel volume for each wash. 9. Fluorography with En3Hance (for 3H and 14C): A. Immerse in minimum amount of En3Hance. Swirl gently for 30 minutes in covered glass dish.

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  • Polyacrylamide Gel Electrophoresis - an overview ...

    Polyacrylamide gel electrophoresis (PAGE) analysis can be conveniently applied to analyze the molecular weight of sulfated GAGs. Gels on which GAGs have been fractionated can be visualized with Alcian Blue with or without silver staining and the bands can be scanned and digitized. The average MW of a GAG is then calculated based on a mixture of HP-derived oligosaccharide …

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  • Section VII: Separation of DNA in Polyacrylamide Gels

    Gel Slick® Solution, increases the ease of separating the glass plates while keeping the gel in place on the other plate for staining — Handling or compression of gels (particularly polyacrylamide-type gels) can lead to regions of high background after staining. If possible, gels should not

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  • 10% Acrylamide Gels for SDS-PAGE

    2008-5-23 · Stacking gel master mix: 340 ml H2O 62.5 ml 1.0 M Tris pH 6.8 5 ml 10% SDS Pouring resolving gel: 1. Make 6 ml of resolving gel (makes 1 gel, with a little bit leftover) 3.96 ml of resolving gel master mix 1.98 ml of 30% acrylamide 60 μl of 10% APS 2.4 μl of TEMED 2. Immediately load gel mixture into the casing with a pipette – fill to the ...

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  • The Nature of Denaturing (Protein Gels, that is!)

    Gelatin zymography. Running the gel. Dilute conditioned media so that all samples have the same protein concentration. F or each sample, test one aliquot at a low protein concentration (5 µg/mL) and one at a high protein concentration (15 µg/mL). Add 5X non-reducing sample buffer to your samples. Prepare a 7.5% acrylamide gel containing gelatin.

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  • Gelatin zymography protocol | Abcam

    2018-8-21 · 9. Run the gel at 5 V/cm, taking care to avoid excessive heating. Run the gel for the time indicated in the certificate of analysis of the ladder. 10. Stain the gel in a 0.5 µg/ml ethidium bromide aqueous solution for about 30 min. Examine the gel under the UV light. This protocol is for the Non-denaturing PAGE

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  • What Are Gradient Gels, Why Use Them, and How to Make

    2001-6-22 · 8. Fix gel. Cut gel down to sample lanes + 1-2 lanes each side. Fix gel in 3 X 5 minute washes of 10% MeOH, 10% AcCOOH (dilute 5X destain 1 in 5). Use ~10 X gel volume for each wash. 9. Fluorography with En3Hance (for 3H and 14C): A. Immerse in minimum amount of En3Hance. Swirl gently for 30 minutes in covered glass dish.

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  • Non-denaturing PAGE - Thermo Fisher Scientific

    2021-3-2 · Principle of SDS PAGE electrophoresis. The techniques polyacrylamide gel electrophoresis is a separating method of protein mixture based on molecular weight. The basic principle of SDS page electrophoresis is the separation based on molecular weight not on shape or charge of molecules.. A uniform charged molecule is migrate in an electric field towards a …

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  • SDS Polyacrylamide Gel Electrophoresis

    2011-7-14 · SDS Polyacrylamide Gel Electrophoresis Gel Recipes % Acrylamide 5% 7.5% 10.% 12.5% 15% 18% 4% Stacking Gel ... Makes ~30.8 ml gel solution for running gel; ~10 ml for stacking gel Electrophoresis Buffer: 5X Buffer: 1 X Buffer 60 g Tris base 9 g Tris base 288 g Glycine 43.2 g Glycine 50 ml 20% SDS 7.5 ml 20% SDS dH 2

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  • The principle and method of polyacrylamide gel ...

    2008-5-23 · Stacking gel master mix: 340 ml H2O 62.5 ml 1.0 M Tris pH 6.8 5 ml 10% SDS Pouring resolving gel: 1. Make 6 ml of resolving gel (makes 1 gel, with a little bit leftover) 3.96 ml of resolving gel master mix 1.98 ml of 30% acrylamide 60 μl of 10% APS 2.4 μl of TEMED 2. Immediately load gel mixture into the casing with a pipette – fill to the ...

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  • Addgene: Protocol - How to Run an Agarose Gel

    2017-8-4 · and lower tank filled with the running buffer is connected by the electrophoresis gel casted in between two glass plates (rectangular and notched). There are additional accessories needed for casting the polyacrylamide gel such as comb (to prepare different well), spacer, gel …

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  • 10% Acrylamide Gels for SDS-PAGE

    2018-8-21 · 9. Run the gel at 5 V/cm, taking care to avoid excessive heating. Run the gel for the time indicated in the certificate of analysis of the ladder. 10. Stain the gel in a 0.5 µg/ml ethidium bromide aqueous solution for about 30 min. Examine the gel under the UV light. This protocol is for the Non-denaturing PAGE

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  • Module 4 Electrophoretic techniques Lecture 21 Basics of ...

    2018-3-2 · The stacking gel solution was prepared to 4% acrylamide, whereas the resolving gel contained 15% acrylamide, 9 M urea at pH 8.9. A Mini PROTEAN cell (Bio-Rad) filled with 0.02 M Tris, 0.19 M Glycine, pH 8.3 running buffer was used. Separation was performed at 4 °C, 0.01 A for 15 min, followed by 0.03 A for 2 h.

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  • Non-denaturing PAGE - Thermo Fisher Scientific

    2007-11-14 · The problem is that the gels seem to run extremely slowly. I'm running a 12% gel right now (SDS-PAGE, 3.5% stacking, 12% sep) and I'm over 1 hour into the run with easily another hour to go, if not longer to go. The sample/dye has just barely made it into the separating gel. I'm running at 100 volts (anything higher and the buffer will be too hot).

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  • Use of urea-polyacrylamide electrophoresis for ...

    2001-6-22 · 8. Fix gel. Cut gel down to sample lanes + 1-2 lanes each side. Fix gel in 3 X 5 minute washes of 10% MeOH, 10% AcCOOH (dilute 5X destain 1 in 5). Use ~10 X gel volume for each wash. 9. Fluorography with En3Hance (for 3H and 14C): A. Immerse in minimum amount of En3Hance. Swirl gently for 30 minutes in covered glass dish.

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  • (PDF) Polyacrylamide Gel Electrophoresis (PAGE) of ...

    Polyacrylamide Gel Electrophoresis (PAGE) of Proteins Brent Turnipseed Professor/Manager, SDSU Seed Testing Lab South Dakota State University Theory of how it works • Electrophoresis is a method where charged molecules in solution, mainly proteins and nucleic acids, migrate in response to an electrical field.

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  • SDS-PAGE Protocol from EnCor Biotechnology Inc.

    2014-4-22 · SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) is probably the worlds most widely used biochemical method. In the early 60's scientists first appreciated the utility of polyacrylamide gels as a convenient and versatile alternative to starch gels Ornstein 1964, Davis 1964 , thus developing polyacrylamide gel electrophoresis or PAGE.

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  • SDS-PAGE PROTOCOL Adapted from Current Protocols, Ch. 10

    2006-2-1 · 1. Prepare polyacrylamide gel according to standard protocol. 2. Load samples and run gel @ 25 mA (2 gels run @ 50 mA) in 1x SDS Running Buffer. 3. At this point, the gel can either be transferred to a membrane (see Western protocol) or stained with Coomassie (see below). 4. Place gel in a plastic container. Cover with isopropanol fixing ...

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  • Polyacrylamide Gel Electrophoresis of RNA

    There are two common types of gel: polyacrylamide and agarose. For most applications, denaturing acrylamide gels are most appropriate. These gels are extremely versatile and can resolve RNAs from ~600 to ≤20 nucleotides (nt). In certain circumstances, e.g., resolving different conformers of RNAs or RNA-protein complexes, native gels are ...

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  • SDS‐Polyacrylamide Gel Electrophoresis (SDS‐PAGE) of ...

    2011-8-1 · Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a technique used to move charged molecules through a gel matrix by means of an electric current. This procedure is used to determine protein subunit composition, verify homogeneity of the protein sample, and purify proteins for use in other applications.

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  • Denaturing Protein Electrophoresis: SDS-PAGE | National ...

    2012-7-23 · For this reason, separation on a polyacrylamide gel in the presence of SDS occurs by mass alone. SDS is the most commonly used detergent in protein electrophoresis. Treatment with SDS creates a uniform charge to mass ratio between different proteins.SDS PAGE offers a rapid and relatively accurate way to determine protein molecular weights ...

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  • SDS PAGE and Western blot - Northern Arizona University

    2011-12-21 · 16. Pour running buffer in the top and bottom chambers, 18. Load the samples in appropriate wells and add more running buffer in the top chamber 20. Run the gel. Use a constant Amps power supply (80 mA). It will take approximately 1-2 hours. 21. Coomassie staining your gel Coomassie stain a. Dissolve 2g Coomassie Blue R, 250 in 250ml water b.

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  • Why are there extra bands visible on polyacrylamide gels ...

    FAQ: Why are there extra bands visible on polyacrylamide gels? To provide increased intensity for the smaller bands and the reference bands, multiple fragments of the same size have been cloned into the plasmids used for many of the DNA ladders. These fragments, identical in size, are indistinguishable on agarose gels, but, on acrylamide gels ...

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  • VERY SLOOOOOW running gels (SDS-PAGE) - Protein and

    2007-11-14 · The problem is that the gels seem to run extremely slowly. I'm running a 12% gel right now (SDS-PAGE, 3.5% stacking, 12% sep) and I'm over 1 hour into the run with easily another hour to go, if not longer to go. The sample/dye has just barely made it into the separating gel. I'm running at 100 volts (anything higher and the buffer will be too hot).

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  • Blue-native PAGE in plants: a tool in analysis of protein ...

    2005-11-16 · Intact protein complexes can be separated by apparent molecular mass using a standard polyacrylamide gel electrophoresis system combining mild detergents and the dye Coomassie Blue. Referring to the blue coloured gel and the gentle method of solubilization yielding native and enzymatically active protein complexes, this technique has been named Blue-Native Polyacrylamide Gel …

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  • SPARQ-ed Risk Assessment Sheet : SDS-Polyacrylamide Gel ...

    SPARQ-ed Risk Assessment Sheet : SDS-Polyacrylamide Gel Electrophoresis Description of Risk Hazard Overall Risk CategoryAnalyse Evaluate Risk Source Current Controls Event Category Consequences Exposure Probability (see explanation on last page) Electrophoresis : High voltages used ~100-200V, risk of electrocution Electricity

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  • The principle and method of polyacrylamide gel ...

    2015-3-17 · Running Buffer, 10X is a Tris-Glycine buffer used for sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE) of proteins. It is used as both the anode and cathode buffer. Running Buffer, 10X is supplied as 1L of 10X concentrate that can be diluted to a 1X solution containing 25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3.

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  • Running Buffer, 10X | SCBT - Santa Cruz Biotechnology

    2012-8-10 · The capacity of a mini-gel for a mixed protein sample is 20 to 40 micrograms/well, depending on the resolution needed and number individual polypeptides in the mix. However, if a pure protein is loaded, one single band will contain all 20-40 micrograms, and the result is a mess.

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  • Smeared protein gels - Rice University

    2021-1-29 · 3. Carefully place the gel in a suitable container such as a polypropylene staining tray. Add a sufficient amount of GelRed® 3X staining solution to submerge the gel. 4. Agitate the gel gently at room temperature for ~30 minutes. 5. Destaining is not required, although the gel can be washed in water to reduce background if necessary. 6.

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  • Revised: January 15, 2021 Product Information

    10 Prepare gels (6 % polyacrylamide; 8M urea) by mixing 60 ml of the urea: acrylamide solution in 10x TBE with 250 µl of ammonium persulfate and 50 µl of TEMED (Sigma). Apply the gel solution to the assembled gel plates (1.5 mm thick) using S2 Sequencing gel electrophoresis apparatus (Invitrogen). Allow the gel to polymerize during 30 min.

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  • Optimization of a reliable, fast, cheap and sensitive ...

    NOTE: Ensure that the gel sandwich at the bottom of the plate is completely soaked in buffer without any bubbles. 4. Running Gels. Load about 1 µL of PCR product into each well of the polyacrylamide gel. Load a DNA size ladder to both sides of the gel along with samples of PCR products. Fix the safety cover to the upper buffer chamber.

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  • A Fast Silver Staining Protocol Enabling Simple and ...

    2011-1-1 · Make a polyacrylamide non-denaturing gel, a 6–8% gel is normally sufficient (see Table 1 for gel recipe). There is a large variety of gel running equipment available on the market (we recommend the Hoefer SE600 CHROMA Vertical Electrophoresis Unit.

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  • Principles and problems of the electrophoretic mobility ...

    2001-6-22 · 8. Fix gel. Cut gel down to sample lanes + 1-2 lanes each side. Fix gel in 3 X 5 minute washes of 10% MeOH, 10% AcCOOH (dilute 5X destain 1 in 5). Use ~10 X gel volume for each wash. 9. Fluorography with En3Hance (for 3H and 14C): A. Immerse in minimum amount of En3Hance. Swirl gently for 30 minutes in covered glass dish.

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  • Denaturing Urea PAGE - Small Gel

    30 seconds. Q. A cell line was engineered to contain and express a DNA sequence frequently associated with pancreatic cancer, with the hope that the cell line will undergo rapid cell division much like the cancerous cells. You want to test whether or not the cell line is efficiently transcribing this DNA by measuring the quantity of mRNA produced.

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