Home | Jobs | News & Articles | Job Advice | Search | Protocols | Fun | @RealLabRat
Lab Rat with DNA

Post your resume
Candidates - post your resume

Search job listings
Candidates - search biotech jobs

Post job openings
Employers - post job openings

Email Login
Password
New users
sign up!
HOME > Protocols > Molecular Biology > DNA > Protocol for DNA Quantitation

Protocol for DNA Quantitation

1. INTRODUCTION

The Turner BioSystems TD-20/20 Luminometer, in combination with Promega’s new DNA Quantitation System, provides a precise and sensitive method for the detection of linear double stranded DNA (dsDNA) including PCR fragments. Plasmid and chromosomal DNA can be quantitated following linearization. The system is unaffected by many of the contaminants that affect other systems. The measurement is based upon a series of coupled enzymatic reactions that produce a light signal proportional to the amount of linear DNA in a sample.

The DNA Quantitation System is more precise than spectrophotometric or agarose gel analyses and is able to detect picogram amounts of DNA. The assay should be used in the linear range of 20pg to 1ng of total DNA added per reaction and can quantitate linear dsDNA species up to 6,000bp in size. Estimation of DNA amount is dependent upon mass and not the number of DNA ends or nicks in the DNA for fragments < 6,000bp.

Since the DNA Quantitation System depends upon the production of ATP by enzymatic reactions, you must take extra care to insure that the samples do not contain dNTPs or NTPs, which can be used to form ATP in the reactions. Unless a DNA sample is contaminant-free, a control not containing T4 DNA Polymerase should be performed. This control reaction will gauge the contribution of signal from materials other than dsDNA. Furthermore, the sample should not contain any ATPase activity.

2. MATERIALS REQUIRED

  • TD-20/20 Luminometer (P/N 2020-000)
  • 8mm Test Tube Adapter (P/N 2020-314)
  • 8mm x 50mm test tubes (P/N 20-015)
  • Microfuge tubes (Sterile)
  • Micropipettes (Aerosol Resistant Tips are Recommended)
  • Sterile 10mM Tris-HCl (pH 7.3-7.5)
  • Sterile TE buffer (pH 7.3-7.5)
  • Sterile deionized water
  • DNA Quantitation System containing:

    2 x 1ml, DNA Quantitation Buffer Solution containing ADP

    2 x 500u, T4 DNA Polymerase (5-10 u/l)

    50 l, Sodium Pyrophohsphate (40mM)

    15l, NDPK Enzyme Solution

    200l, DNA Quantitation DNA Standard (5ng/l 5%)

    1 vial, ENLITEN Luciferase/Luciferin reagent

    1 vial, ENLITEN Reconstitution Buffer (12ml)

3. EXPERIMENT PROTOCOL

3.1 ENLITEN Luciferase/Luciferin reagent preparation: Use as supplied by Promega Corporation. Store at -20 C. Lightly tap the vial of ENLITEN L/L Reagent before opening to gather all of the dried material to the bottom of the vial. Transfer 12ml of the ENLITEN L/L Reconstitution Buffer into the vial of ENLITEN L/L Reagent. Replace the stopper carefully, and gently invert the vial several times to dissolve the contents. Do not shake the dissolved ENLITEN L/L Reagent. Equilibrate the reconstituted ENLITEN L/L Reagent to room temperature for a minimum of 30 minutes and a maximum of 8 hours. Unused reconstituted reagent should be dispensed into sterile microcentrifuge tubes and frozen at -20 C

3.2 Reaction Master Mix Preparation: Thaw the enzyme and buffer solution and keep on ice. Mix the reagents well by gentle inversion or vortexing.

Note: Prepare the reaction master mix just prior to use. Determine the number of reactions to be performed for samples and that to prepare a standard curve. All samples and controls should be performed in duplicate or triplicate.

Prepare a master mix using Table 1 as a guide. Add the components in the order listed to a fresh microcentrifuge tube, mix gently and keep the solution on ice.

Number of Reactions (n)

Component

1–9

10

Experiment

DNA Quantitation
Buffer Solution

n 15.5l

n 15.5l

_ __l = ___l

Sodium Pyrophosphate

n 0.5l

n 0.5l

_ __l = ___l

NDPK Enzyme Solution

n 1.0l

n 0.1l

_ __l = ___l

Water, sterile deionized

n 0.9l

_ __l = ___l

T4 DNA Polymerase

n 1.0l

n 1.0l

_ __l = ___ l

Total Volume

n 18.0l

n 18.0l

_ __l = ___l

 

Table 1. Volume of Master Mix Components Required by Number of Reactions.

4. INSTRUMENT SET-UP

4.1 Turn on instrument and allow to warm up for at least 5 minutes (600 seconds).

4.2 Adjust settings so that:

Delay period = 3sec.
Integrate period = 15sec.
Replicates = 1

5. SAMPLE ANALYSIS

5.1 Prepare the reconstituted ENLITEN L/L Reagent. Allow to equilibrate to room temperature.

5.2 Prepare samples for the standard curve by making dilutions of the provided DNA Quantitation DNA Standard. The DNA standard should be mixed well. Dilutions can be made using 10mM Tris-HCl (pH 7.3–7.5) or TE buffer (10mM Tris, 1mM EDTA [pH 7.3–7.5]). The prepared samples should span the region of interest in the linear range of the curve.

5.3 If needed, dilute the DNA samples to be tested to 10–500pg/l using 10mM Tris-HCl (pH 7.3–7.5) or TE buffer.

5.4 Prepare the reaction master mix.

5.5 Aliquot 18l of master mix into each prelabeled microcentrifuge tube.

5.6 Add 2l of DNA solution (from duplicate or triplicate reactions) to each of the tubes and vortex gently. Include a negative control containing 2l of the buffer used to dilute the standard.

5.7 Incubate at 37C (in a heating block or water bath) for 8–10 minutes. Do not incubate longer.

5.8 Place samples on ice and analyze as soon as possible.

5.9 Remove 15l from one reaction mix and place into 100l of reconstituted ENLITEN L/L Reagent. Mix gently.

5.10 Immediately read the sample by pressing "GO" on the TD-20/20. Repeat Steps 9 and 10 until all samples are read.

6. DNA CONCENTRATION CALCULATION

6.1 Average duplicate or triplicate sample values.

6.2 Subtract the averaged value for the "no DNA" blank reaction from that obtained for the other samples. This yields net light units (LU).

Net average LU (y) = Average LU sample – Average LU No DNA Control

6.3 Generate the standard curve by plotting DNA concentration vs. net light units and drawing a best-fit line (of the form y = mx, or y = mx + b).

6.4 Determine the concentration of the unknown samples by comparing the net light units seen for these samples against those seen for the standard curve.

7. RECOMMENDED READING

"DNA Quantitation System," Technical Bulletin 278 (available from Promega Corporation).

theLabRat.com 2005. All Rights Reserved.