For general laboratory use. FOR IN VITRO USE ONLY.

DIG Luminescent Detection KitDetection of digoxigenin-labeled nucleic acids by enzyme immunoassay with luminescence on nylon membranes.

Cat. No. 1 363 514Kit for the detection of 50 blots of 100 cm² Store at �15 to �25° C

Instruction ManualVersion 2, March 2001

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1. Preface

1.1 Table of contents

1. Preface .....................................................................................................................................................31.1 Table of contents ................................................................................................................................................................. 31.2 Kit contents ............................................................................................................................................................................ 4

2. Product overview ...................................................................................................................................5

3. Procedures and required materials ...................................................................................................83.1 Immunological detection with CSPD ............................................................................................................................ 83.2 Stripping and reprobing of DNA blots .......................................................................................................................10

4. Results ...................................................................................................................................................11

5. Appendix ................................................................................................................................................125.1 Trouble shooting ................................................................................................................................................................125.2 How to contact Roche Molecular Biochemicals .....................................................................................................135.3 References ............................................................................................................................................................................135.4 Related products ................................................................................................................................................................13

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1.2 Kit contents

Bottle/Cap

Label Contentincluding function

Cat. No.

1 Labeled control DNA

50 �l linearized pBR328 DNA, labeled with digoxigenin according to the standard protocol containing 1 pg template DNA and approx. 260 ng synthesized labeled DNA.

1 585 738

2 DNA dilution buffer

1 ml herring sperm DNA (50 pg/ml) in 10 mM Tris-HCl, 1 mM EDTA, pH 8.0 (20° C).

3 Anti-digoxi-genin-AP, Fab fragments

100 �l polyclonal sheep anti -dig-oxigenin, Fab fragments, conjugated to alkaline phosphatase

1 633 716

4 Blocking reagent Two bottles with 50 g powder each 1 096 176

5 CSPD1) 1 ml CSPD Disodium 3-(4-methoxy-spirofl,2-dioxetane-3,2'-(5'-chloro) tricyclo [3.3.l.l3,7]decan)4-yl) phenyl phosphate, molecular weight: 46,1.

1 655 884

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2. Product overview

Test principle The nonradioactive DIG system uses digoxigenin, a steroid hapten, coupled to dUTP, UTP or ddUTP to label DNA, RNA or oligonucleotides for hybridization and subsequent luminescent detection (1-4).

Fig. 1:

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DIG-labeling reaction

Please find in the following table the labeling techniques for DNA, RNA, and oligonu-cleotides.

Application Highly sensitive detection of DIG-labeled nucleic acids on all types of membrane blots, using anti-digoxigenin, alkaline phosphatase conjugates and the chemiluminescent substrate CSPD.

Immunological detection

The hybridized probes are immunodetected with anti-digoxigenin, Fab fragments conju-gated to alkaline phosphatase and are then visualized with the chemiluminescence substrate CSPD. Enzymatic dephosphorylation of CSPD by alkaline phosphatase leads to a light emission at a maximum wavelength of 477 nm which is recorded on X-ray films.The chemiluminescent signal from CSPD persist for days on nylon membranes. Since film exposures of a few minutes are usually sufficient for signal detection, multiple images may be acquired.(Fig. 2)

Fig. 2

Sample material DIG labeled nucleic acids.

Assay time

Nucleic acid Labeling reaction

DNA probes Labeling with DIG-dUTP via random-primed labeling, nick translation or PCR.

Oligonucleotides 3’-end labeling with DIG-ddUTP or tailed with DIG-dUTP using terminal transferase.5’-end labeling of oligonucleotides is done with DIG-NHS-ester (digoxigenin-3-O-methylcarbonyl-�-aminocaproic-acid-N-hydroxysuccinimide ester).

RNA probes Synthesis in an in vitro transcription reaction with SP6, T7 or T3 RNA polymerases.

O OOCH3

OPO3Cl

CSPD

2-

alkalinephosphatase

O OOCH3

OCl Cl

OOCH3

metastableintermed iate

CSD

+

h v

O

O*

Step Reaction time

Immunological detection 1.5 h

Signal detection 5 - 30 min

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Number of detections

50 blots with a size of 10 × 10 cm.

Quality control Each lot of CSPD is tested for purity: CSPD (NMR) > 98%Using DIG-labeled control DNA (pBR328/Bam HI) as hybridization probe, 0.03 pg homologous DNA diluted with 50 ng heterologous DNA are detected in a dot blot with CSPD after � 30 min exposure to X-ray film, following the standard detection protocol.

Kit storage/ stability

The unopened kit is stable at �15 to �25° C through the expiration date printed on the label.All components of the kit are stable at �15 to �25° C.In the following table storage and stability instructions for several kit components are listed:

Sensitivity and specificity

A single copy gene (tissue plasminogen activator, tPA) is detected in a Southern blot of 0.3 pg Bgl II or Eco RI digested human placenta DNA. The same sensitivity is obtained, when using DIG-labeled RNA probes

Reagent Storage/Stability

Antibody conjugate (vial 3) once opened, should be stored at 2-8° C

Blocking reagent (bottle 4) dry at 2-8° C or at 15-25° C

CSPD (vial 5) • at 2-8° C when frequently used.• Repeated freeze/thaw cycles should be avoided.

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3. Procedures and required materials

3.1 Immunological detection with CSPD

Time requirements

In the following table the time requirements for the single steps are listed:

Additional equipment and reagents required

• Hybridization bags (Cat. No. 1 666 649)or

• Development folders• plastic or glass boxes or petri dishes • DIG Wash and Block Buffer Set (Cat. No. 1 585 762)

or• Washing buffer• Maleic acid buffer• Detection buffer

Preparation of additional solutions required

The Washing buffer, Maleic acid buffer, and Detection buffer are also available in the DIG Wash and Block Buffer Set (Cat. No. 1 585 762).

Step Time

Washing and Blocking of membrane 32 min

Antibody binding 30 min

Washing and equilibration of membrane 32 min

Luminescent reaction 5 min

Preincubation at 37°C 10 min

Film exposure 20 min

Total time 130 min

Solution Composition / Preparation Storage/stability

Use

Washing buffer

0.1 M Maleic acid, 0.15 M NaCl; pH 7.5 (20° C); 0.3% (v/v) Tween2) 20

15-25° C, stable

Removal of unbound antibody

Maleic acid buffer

0.1 M Maleic acid, 0.15 M NaCl; adjust with NaOH (solid) to pH 7.5 (20° C)

15-25° C, stable

Dilution of Blocking solution

Detection buffer

0.1 M Tris-HCl, 0.1 M NaCl, pH 9.5 (20° C)

15-25° C, stable

Adjustment of pH to 9.5

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Preparation of kit working solutions

Note: Please refer to the following table:

Procedure All incubations should be performed at 15-25° C with agitation. The volumes are calculated for a membrane size of 100 cm2.

Solution Composition / Preparation Storage/stability

Use

CSPD(vial 5)

Dilute CSPD 1:100 in Detection buffer. [0.1 M Tris-HCl, 0.1 M NaCl, pH 9.5 (20° C)].Note: The solution can be reused one to two times.

2-8° C in the dark

Chemilumines-cence detection

Antibody solution(vial 3)

Centrifuge Anti-Digoxigenin-AP (vial 3) for 5 min at 10 000 rpm in the original vial prior to each use, and pipet the necessary amount carefully from the surface. Dilute Anti-Digoxigenin-AP 1:10 000 (75 mU/ml) in Blocking solution.

2-8° C Binding to the DIG-labeled probe

Blocking stock solution (10 × conc.)(bottle 4)

Dissolve Blocking reagent 10%(w/v) in Maleic acid buffer under constantly stirring on a heating block (65°C) or heat in a microwave oven, autoclave. The solution remains opaque.

2-8° C Preparation of Blocking solution

Blocking solution

Prepare a 1 × working solution by diluting the 10 × Blocking solution 1:10 in Maleic acid buffer.

Always prepare

fresh

Blocking of unspecific binding sites on the membrane

Step Action1 After hybridization and stringency washes, rinse membrane briefly (1-5) min in

Washing buffer.2 Incubate for 30 min in 100 ml Blocking solution.3 Incubate for 30 min in 20 ml Antibody solution.4 Wash 2 × 15 min in 100 ml Washing buffer.5 Equilibrate 2-5 min in 20 ml Detection buffer.6 • Place membrane with DNA side facing up on a development folder

(or hybridization bag) and apply 2 ml diluted CSPD solution.• Immediately cover the membrane with the second sheet of the folder to

spread the substrate evenly and without airbubbles over the membrane.• Incubate for 5 min at 15-25° C.

7 Squeeze out excess liquid and seal the edges of the development folder.8 Incubate the damp membrane for 5-15 min at 37° C to enhance the luminescent

reaction.9 Expose to Lumi-Imager3) for 5-20 min or to X-ray film for 15-25 min at 15-25° C.

Note: Luminescence continues for at least 24 hours and signal intensity remains almost constant during the first hours.Multiple exposures can be taken to achieve the desired signal strength.

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3.2 Stripping and reprobing of DNA blots

General The alkali-labile form of DIG-11-dUTP enables easier and more efficient stripping of blots for rehybridization experiment.Note: If filters are to be stripped and reprobed, they should not be allowed to dry out, but should be stored in 2 x SSC or maleic acid.

Additional equipment and reagents required

• Large beaker• Water bath• 10 × SSC• 10% SDS• 0.2N NaOH

Procedure Please refer to the following table.Note: Alternative stripping protocols, as mentioned in the "DIG System User´s Guide for Filter Hybridization" (available on request) can also be used with high efficiency.

Step Action

1 Rinse membrane thoroughly in double distilled water.

2 Wash for 2 × 15 min at 37° C in 0.2 M NaOH containing 0.1% SDS to remove the DIG-labeled probe.

3 Rinse thoroughly 5 min in 2 × SSC.

4 Prehybridize and hybridize with a second probe.

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4. Results

Detection of DIG labeled Nucleic Acids with CSPD

Fig. 3

Human genomic DNA was digested with Eco RI, separated on a 1% agarose gel, and blotted onto positively charged nylon membranes. The blots were hybridized with 50 ng/ml DIG-labeled β-actin RNA. Chemiluminescent detection was according to the standard DIG chemiluminescent detection procedure using CSPD at 0.25 mM final concentration.

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5. Appendix

5.1 Trouble shooting

Trouble shooting table

This table describes various troubleshooting parameters for DIG-labeling and detection

Problem Possible cause Recommendation

Low sensitivity Inefficient probe labeling

• Check labeling efficiency. The labeling reaction can be upscaled. Prolong incubation time to overnight.

• Clean up template DNA by phenolization.• Use only fragments � 10 kb or predigest with a

restriction enzyme (e.g., a four bp cutter).• Check the amount and quality of target DNA.• Make sure that template is efficiently denatured

before labeling.

Low probe con-centration in the hybridization

• Increase probe concentration (use 25 ng/ml).• Prolong hybridization time to overnight.• Increase concentration of anti-DIG-AP (dilute

1:5000)

High back-ground

Inefficient hybridization

• Recalculate hybridization temperature.• Do not allow the membrane to dry between

prehybridization and hybridization.• If you use plastic bags, remove all air bubbles

prior to sealing.

• Use DIG Easy Hyb* buffer, especially when other membrane brands are used.

Concentration of labeled probe is too high

Determine optimal probe concentration as described in section 3.1, do not use more than 25 ng/ml.a) Decrease probe concentrationb) Increase volume of prehybridization solution.

Wrong type of nylon membrane

Some types of nylon membrane may cause high background, use Roche Biochemicals nylon mem-brane, especially tested for the DIG-System.

Inefficient block-ing before immuno-assay

Prolong blocking and washing steps.

When using laboratory trays for the detection proce-dure, they should be rigorously cleaned before use. Anti-DIG-AP binding and chemiluminescent de-velopment should be done; in separate trays.

Heat treatment of all glass ware is recommended to solve background problems

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5.2 How to contact Roche

3 ways to contact us

To contact Roche Molecular Biochemicals for technical assistance, please choose one of the following:

5.3 References

1 Höltke, H. J. et al. (1995) Cellular and Molecular Biology 41(7), 883-905.2 Bronstein, I. et al. (1991) in Bioluminescence and Chemiluminescence, Current Statur (Stanley, P. & Kricka, L.J., eds)

pp 73-82.3 Schaap, A. P. et al. (1989) Clin. Chem. 35, 1863

5.4 Related products

Kits

IF you are using... THEN...

the Internet access our web-site at:http://biochem.roche.com

E-mail identify the address which corresponds to your particular location, printed on the last page of this instruction manual.

telephone please refer to the telephone number which corresponds to your particular location, printed on the back cover of this instruction manual.

Product Pack Size Cat. No

DIG DNA Labeling Kit 40 labeling reactions 1 175 033

DIG Oligonucleotide 3’-End Labeling Kit

25 reactions 1 362 372

DIG Oligonucleotide Tailing Kit 25 reactions 1 417 231

DIG- RNA Labeling Kit (SP6/T7) 2 × 10 reactions 1 175 025

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Single reagents

Product Pack Size Cat. No.

Anti-digoxigenin-AP, Fab fragments 50 U 1 633 716

Blocking reagent 50 g 1 096 176

CDP-Star4) 1 ml2 ml

1 685 6271 759 051

CDP-Star, ready-to-use 2 × 50 ml 2 041 677

CSPD1) 1 ml2 ml4 ml

1 655 8841 759 0351 759 043

CSPD, ready-to-use 2 × 50 ml 1 755 633

DIG Easy Hyb 500 ml 1 603 558

DIG Easy Hyb Granules Granules for 6 × 100 ml 1 796 895

DIG RNA Labeling Mix 40 �l (20 reactions) 1 277 073

DIG Wash and Block Buffer Set 30 blots (10 × 10 cm) 1 585 762

DIG-11-ddUTP 25 nmol (25 �l) 1 363 905

DIG-11-dUTP, alkali-labile 25 nmol (25 �l)125 nmol (125 �l)

1 573 1521 573 179

DIG-11-UTP 250 nmol (25 �l) 1 209 256

DIG-3'-end labeled control oligonu-cleotide

50 �l (125 pmol) 1 585 754

DIG-DNA Labeling Mix 50 �l (25 reactions) 1 277 065

DIG-High Prime 160 �l (40 reactions) 1 585 606

DIG-labeled control DNA 50 �l 1 585 738

DIG-labeled control RNA 50 �l 1 585 746

DIG-NHS Ester Digoxigenin-3-0-methyl-carbonyl-�-amino-caproic acid-N-hydrox-ysuccinimide ester

5 mg 1 333 054

DNA, MB grade 500 mg (50 ml) 1 467 140

Hybridization Bags 50 bags 1 666 649

Lumi-Film 100 sheets (8 × 10 cm)100 sheets(18 × 24 cm)100 sheets (35 × 43 cm)

1 666 6571 666 9161 666 711

Lumi-Imager F13) 240 Volt120 Volt

2 015 1702 012 847

Nylon membranes, positively charged

10 sheets (20 × 30 cm)20 sheets (10 × 15 cm)

1 roll (0.3 × 3m)

1 209 2991 209 2721 471 240

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1) CSDP is a trademark of Tropix Inc., Bedford, MA, USA.2) Tween is a trademark of ICI Americas, Inc., USA.3) Lumi-Imager is a trademark of a Member of the Roche Group.4) CDP-Star is a trademark of Tropix Inc., Bedford, MA, USA.

Sold through an arrangement with ENZO DIAGNOSTICS, INC. Purchase of this product does not include any right or license to exploit this product commercially.This product or the use may be covered by one or more claims of ENZO patents, including but not limited to the following: U.S. Patent Nos. 5,328,824; 5,449,767; 5,476,928; 4,994,373; 5,175,269; and 5,002,885; EP 0 063 879 BI; EP 0 329 198 B1; EP 0 122 614 BI; EP 0 128 332 BI; DK 171 822; Canadian Patent Nos. 1,219,824; 1,254,525; 1,309,672; and 1,228,811; Japanese Patent Nos. 2,131,226; 1,416,584; 2,595,201; 2,577,881; and patents pending.This product or the use of this product may be covered by one or more patents of Roche Molecular Biochemicals, including the following:EP patent 0324 474 (granted)U.S. patent 5.354.657 (granted)

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