For life science research only. Not for use in diagnostic procedures. FOR IN VITRO USE ONLY.

DIG DNA Labeling and Detection Kit

Random primed DNA labeling with digoxigenin-dUTP, alkali-labile and detection of hybrids by enzyme immunoassay

Cat. No. 11 093 657 910 Kit for 25 labeling reactions of 10 ng-3 g DNA and detection of 50 blots of 100 cm Store at

15 to

25 C

Instruction Manual
Version October 2004

1.
1.1 1. 1.1 1.2 2. 2.1 3. 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 4. 4.1 5. 5.1 5.2 5.3

Preface
Table of Contents Preface .....................................................................................................................................................2 Table of Contents ................................................................................................................................................................. 2 Kit contents ............................................................................................................................................................................ 3 Introduction ............................................................................................................................................5 Product overview .................................................................................................................................................................. 5 Procedures and required materials ...................................................................................................7 Before you begin .................................................................................................................................................................. 7 Flow chart ............................................................................................................................................................................... 8 DIG-DNA Labeling .............................................................................................................................................................. 8 Determination of labeling efficiency ...........................................................................................................................10 DNA transfer and fixation ...............................................................................................................................................13 Hybridization ........................................................................................................................................................................13 Immunological detection ................................................................................................................................................15 Stripping and reprobing of DNA blots .......................................................................................................................17 Results ...................................................................................................................................................18 Typical results .....................................................................................................................................................................18 Appendix ................................................................................................................................................19 Trouble shooting ................................................................................................................................................................19 References ............................................................................................................................................................................20 Ordering Information .........................................................................................................................................................20

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1.2

Kit contents Bottle/ Label Content Cap including function 1 Unlabeled control 20 l pBR328 DNA 1 [100 g/ml] pBR328 is digested separately with Bam HI, Bgl I and Hinf I. The digests are combined in a ratio of 2:3:3. Sizes of the 16 pBR328 fragments: 4907; 2176; 1766; 1230; 1033; 653; 517; 453; 394; 298 (2 ); 220 and 154 (2 ) bp. clear solution control DNA for Southern blot 2 Unlabeled control 20 l pBR328 DNA 2 [200 g/ml] Linearized with Bam HI clear solution control for labeling reaction 3 DNA Dilution 2 1 ml Buffer [50 g/ml herring sperm DNA in 10 mM Tris-HCl, 1 mM EDTA; pH 8.0 at +20C] clear solution 4 Labeled Control 50 l pBR328 DNA linearized with Bam HI DNA [5 g/ml] clear solution determination of labeling efficiency 5 Hexanucleotide 50 l Mix 10 concentrated hexanucleotide reaction mix clear solution component of the labeling reaction 6 dNTP labeling 50 l mixture 10 concentrated dNTP labeling mix containing: 1 mM dATP, 1 mM dGTP, 1 mM dCTP, 0.65 mM dTTP, 0.35 mM DIG-11-dUTP pH 7.5 (20 C) clear solution component of the labeling reaction 7 Klenow enzyme, 25 l Klenow enzyme, labeling grade labeling grade 2 U/ l clear solution enzym for the DNA synthesis 200 l 8 AntiDigoxigenin-AP [750 U/ml] Conjugate polyclonal sheep anti-digoxigenin, Fab-fragments, conjugated to alkaline phosphatase clear solution Continued on next page

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1.2

Kit contents, Continued Bottle/ Label Cap 9 NBT/BCIP Content including function 10 1 ml concentrated stock solution can vary between light yellow and brown, clear solution Reacts with alkaline phosphatase. 2 bottles with 50 g powder each

10

Blocking reagent

Additional In addition to the reagents listed above, you have to prepare several solutions. In the equipment and table you will find an overview about the equipment which is needed for the different reagents required procedures. Detailed information is given in front of each procedure. Procedure 3.3 DIG-DNA labeling 3.4 Determination of labeling efficiency Equipment water bath Nylon membranes positively charged* Reagents sterile, double distilled water EDTA, 0.2 M, pH 8.0, sterile DIG Wash and Block Buffer Set* TE-buffer or Washing buffer Maleic acid buffer Detection buffer TE-buffer 2 SSC or 10 SSC DIG Easy Hyb *

3.5 DNA transfer and fixation 3.6 Hybridization

UV- light box or commercial available UV-cross linker

3.7 Immunological detection

Nylon membranes, positively charged* Hybridization bags* or temperature resistant, sealable plastic bags or roller bottles Note: Do not use open trays when working with DIG Easy Hyb buffer temperature resistant plastic bags or DIG Wash and Block Buffer roller bottles Set*TE-buffer Hybridization bags* or Washing buffer Maleic acid buffer Detection buffer TE-buffer

Continued on next page

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Additional equipment and reagents required,

Continued

Procedure Equipment 3.8 Stripping and reprobing Large tray of DNA blots Water bath

Reagents Dimethylformamid (DMF) 0.2 N NaOH, 0.1% SDS (w/v) 2 SSC

2.
2.1

Introduction
Product overview DIG-labeled DNA probes are generated according to the method of random primed labeling [1] which is based on the hybridization of random oligonucleotides to the denatured DNA template. The complementary DNA strand is synthesized by Klenow enzyme which uses the [3`OH] termini of the random oligonucleotides as primers and a mixture of deoxyribonucleotides containing DIG-11-dUTP, alkali-labile for elongation. DIG dUTP is incorporated every 20-25 nucleotides into the newly synthesized DNA. This density of haptens in the DNA yields the highest sensitivity in the detection reaction. The DIG DNA Labeling and Detection Kit uses digoxigenin (DIG), a steroid hapten, to label DNA probes for hybridization and subsequent color detection by enzyme immunoassay. Stage DNA labeling Hybridization Description DIG-labeled DNA probes are generated according to the random primed labeling technique. DIG-labeled probes are used for hybridization to membrane blotted nucleic acids according to standard methods. The use of the alkali-labile form of DIG-11-dUTP enables easier and more efficient stripping of blots for rehybridization with a second DIG-labeled probe. The hybridized probes are immunodetected with anti-digoxigeninAP, Fab fragments and are then visualized with the colorimetric substrates NBT/BCIP.

Labeling principle

Test principle

Immunological detection

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Fig. 1: The figure shows DIG-11-dUTP.

Application Sample material

DIG-labeled DNA probes can be used for all types of filter hybridization. DNA fragments of at least 100 bp linearized plasmid, cosmid or DNA supercoiled DNA This table lists the reaction time of the single steps Step DNA labeling Hybridization Immunological detection Color development Reaction time 1 h-O/N 6 h or O/N 1.5 h 0.5-16 h

Assay time

Number of tests

1 kit is sufficient for 25 standard labeling reactions of up to 3 g template DNA and detection of 50 blots of 100 cm2. Using unlabeled control-DNA (pBR328), labeled as described in the protocol, 0.1 pg of homologous DNA is detected in a dot blot after 16 h color development (1 pg of homologous DNA can be detected after 1h color development).

Quality Control

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Kit storage/ stability

The unopened kit is stable at 15 to 25 C until the expiration date printed on the label. Shipping conditions on dry ice. Once opened, please refer to the following table for proper storage. Kit component Anti-Digoxigenin-AP Conjugate vial 8 NBT/BCIP vial 9 Storage 2-8 C, stable 2-8 C, stable or at least 4 weeks at 15-25 C Note: During shipment of the kit on dry ice, a precipitate may occur which is easily dissolved by briefly warming to 37 C dry at 2-8 C or at 15-25 C. as 10 solution aliquot and store at 15 to 25 C, or at 2-8 C until one month under sterile conditions. the working solution should be always freshly prepared.

Blocking reagent (bottle 10)

Sensitivity and specificity

A single copy human gene is detected in a Southern blot of 1 g digested placenta DNA. Note: Sensitivity depends both on the concentration of labeled DNA in the hybridization and on the time of color reaction.

3.
3.1

Procedures and required materials

Before you begin

General handling This table describes general hints for DIG labeling and detection. recommendations Recommendation Guideline Work under clean conditions Autoclave DIG System solutions Filter-sterilize solutions containing SDS Tween1) 20 should be added to previously sterilized solutions Use clean incubation trays Rigorously clean and rinse laboratory trays before each use. Membrane handling requirements Wear powder-free gloves Handle membrane only at the edges and with clean forceps

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Flow chart

Section 3.3 Section 3.4 Section 3.5 Section 3.6 Section 3.7 Section 3.8

DIG-DNA labeling Quantification of labeling efficiency DNA fixation Hybridization Immunological detection Stripping and reprobing of DNA blots

3.3

DIG-DNA Labeling DNA is random primed labeled with Digoxigenin-11-dUTP using a mixture of random hexamers, a dNTP mix containing alkali-labile Digoxigenin-11-dUTP and labeling grade Klenow enzyme.

Introduction

water bath Additional equipment and ice/water reagents required This table lists composition, storage and use of the required reagents in addition to kit components Solution Water EDTA Composition Autoclaved, double distilled water 0.2 M ethylene-diamino- tetracetic acid, pH 8.0 Storage Use 15-25 C, Dilution of DNA stable 15-25 C, Stopping the stable labeling reaction

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Template DNA

The following table lists the recommended features of the template DNA Feature Purity Detail For plasmid DNA use the High Pure Plasmid Isolation Kit * for purification. When other commercially available purification kits are used, we recommend to do an additional phenol/chloroform extraction to remove residual protein. This step is also necessary when templates have been treated with restriction or other modifying enzymes before labeling. To obtain optimal results, template DNA should be linearized and should have a size of 100-10 000 bp or larger. Template DNA 10 kb should be restriction-digested using a 4 bp cutter (e.g. Hae III) prior to labeling. With the procedure described below 10 ng3 g of template can be labeled. By scaling up of all volumes and components accordingly, this procedure can be used for labeling of larger amounts. If singlecopy gene detection in complex genomes is performed at least 300 ng of template DNA (probe concentration: 25 ng/ml hybridization solution) should be labeled.

Size

Amount

Procedure

In the following table please find a protocol for the standard labeling assay. Note: Larger amounts can be labeled by scaling up of all components and volumes. Linear DNA is labeled more efficiently than circular and supercoiled DNA. Step 1 Action Add 10 ng - 3 g DNA and autoclaved, double distilled water to a final volume of 15 l to a reaction vial. For a control labeling reaction use 5 l of control DNA 2 (vial 2) and add 10 l double distilled water. Denature the DNA by heating in a boiling water bath for 10 min and quickly chilling in an ice/water bath. Note: Complete denaturation is essential for efficient labeling. Add the following to the freshly denatured probe or control DNA. Reagent Hexanucleotide Mix, 10 (vial 5) dNTP Labeling Mix (vial 6) Klenow enzyme labeling grade (vial 7) Volume 2 l 2 l 1 l

Mix and centrifuge briefly. Incubate for 1 h to 20 h (overnight) at 37 C. Note: Longer incubation (up to 20 h) will increase the yield of labeled DNA (see table below). Stop the reaction by adding 2 l 0.2 M EDTA (pH 8.0) and/or by heating to 65 C for 10 min. Note: The length of the DIG-labeled fragments range from 200 to 1000 bp.

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Yield of labeling reaction

Table 1: Labeling reactions were performed with increasing amounts of different template DNAs for 1 h and 20 h. The yield of DIG-labeled DNA was determined by incorporation of a radioactive tracer and confirmed by a dot blot (Average of 10 independent labeling assays). Template DNA 10 ng 30 ng 100 ng 300 ng 1000 ng 3000 ng 1h 15 ng 30 ng 60 ng 120 ng 260 ng 530 ng 20 h 50 ng 120 ng 260 ng 500 ng 780 ng 890 ng

Labeling of DNA isolated from agarose

For hybridization of genomic Southern blots, you should separate the template insert DNA from the vector by agarose gel electrophoresis. To isolate DNA from the gel, you can use the Agarose Gel DNA Extraction Kit* for DNA fragments in the range of 400 bp to 5 kbp. It is applicable for standard agarose gels as well as low melting point agarose gels. Afterwards, the DNA fragments are efficiently labeled with digoxigenin without further purification. However, labeled probes should be purified with the High Pure PCR Product Purification Kit* to remove residual agarose particles.

3.4

Determination of labeling efficiency Determination of the yield of DIG-labeled DNA is most important for optimal and reproducible hybridization results. Too high of a probe concentration in the hybridization mix causes background, while too low of a concentration leads to weak signals. The preferred method for quantification of labeled probes is the direct detection method. Stage 1 Description A series of dilutions of DIG-labeled DNA is applied to a small strip of nylon membrane positively charged*. Part of the nylon membrane is preloaded with defined dilutions of DIG-labeled control DNA (vial 4) which are used as standards. The nylon membrane is subjected to immunological detection with anti-digoxigenin-AP conjugate (vial 8) and the freshly prepared color-substrate solution. The color intensities of the dilution series of DIG-labeled DNA and control DNA are compared and amounts calculated.

Introduction

Test principle

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Preparation of additional solutions required

Please find in the following table composition and preparation of additional reagents required. The following buffers are also available in the DIG Wash and Block Buffer Set (Cat. No. 1 585 762) DNase and RNase free. Solution Composition / Preparation Storage and stability 15-25 C, stable 15-25 C, stable 15-25 C, stable 15-25 C, stable Use

Washing buffer Maleic acid buffer Detection buffer TE-buffer

0.1 M Maleic acid, 0.15 M NaCl; pH 7.5 (20 C); 0.3% (v/v) Tween 20 0.1 M Maleic acid, 0.15 M NaCl; adjust with NaOH (solid) to pH 7.5 (20 C) 0.1 M Tris-HCl, 0.1 M NaCl, pH 9.5 (20 C) 10 mM Tris-HCl, 1 mM EDTA, pH 8.0

Removal of unbound antibody Dilution of Blocking solution Adjustment of pH to 9.5 Stopping color reaction

Preparation of kit The following table shows the preparation of kit working solutions working solutions Solution Composition/ Preparation Storage and stability Blocking Dissolve Blocking reagent (bottle 10) 2-8 C stock 10% (w/v) in Maleic acid buffer under solution constantly stirring on a heating block (10 conc.) (65C) or heat in a microwave oven, autoclave. The solution remains opaque. Blocking Prepare a 1 working solution by dilutAlways solution ing the 10 Blocking solution 1:10 in prepare Maleic acid buffer. fresh Antibody solution Centrifuge Anti-Digoxigenin-AP (vial 8) 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 AntiDigoxigenin-AP 1: 5 000 (150 mU/ml) in Blocking solution. Add 40 l of NBT/BCIP (vial 9) to 2 ml of Detection buffer. Note: Store protected from light! 12 h at 2-8 C

Use

Preparation of Blocking solution

Blocking of unspecific binding sites on the membrane Binding to the DIG-labeled probe

Colorsubstrate solution

always prepare fresh

Visualization of antibody-binding

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Dilution series

Labeled probes and the DIG-labeled control DNA (vial 4) must be diluted to 1 ng/ l, according to the expected yield of synthesized nucleic acid to start the dilution series below. The expected yield of DIG-labeled DNA in your probe can best be estimated by using the chart in chapter 3.3. The yield depends on the starting amount of template and incubation time. Note: The yields given in table 1 were achieved under optimal conditions with highly purified template DNA. Tube DNA ( l) 1 2 3 4 5 6 7 8 9 5 15 5 5 5 5 5 0 From tube # original 1 2 2 3 4 5 6 DNA Dilution Buffer (vial 3) ( l) 495 35 45 45 45 45 45 50 Dilution 1:100 1:3.3 1:10 1:10 1:10 1:10 1:10 Final concentration 1 ng/ l 10 pg/ l 3 pg/ l 1 pg/ l 0.3 pg/ l 0.1 pg/ l 0.03 pg/ l 0.01 pg/ l 0

Procedure

The following procedure describes the direct detection. Note: Use sufficient buffer volumes to cover the membrane completely during all steps. Step Action 1 Apply a 1 l spot of tubes 2-9 from your labeled probes and the labeled control to a small strip of nylon membrane. 2 Fix the nucleic acid to the membrane by cross linking with UV-light or baking for 30 min at 120 C. 3 Transfer the membrane into a plastic container with 20 ml Maleic acid buffer. Incubate under shaking for 2 min at 15-25 C. 4 Incubate for 30 min in 10 ml Blocking solution. 5 Incubate for 30 min in 10 ml Antibody solution. 6 Wash with 10 ml Washing buffer, 2 15 min. 7 Equilibrate 2-5 min in 10 ml Detection buffer. 8 Incubate membrane in 2 ml freshly prepared Colorsubstrate solution in a appropriate container in the dark. Do not shake during color development. Note: The membrane can be exposed to light for short time periods to monitor color development. 9 Stop the reaction, when desired spot intensities are achieved, by washing the membrane for 5 min with sterile double dist. water or with TE-buffer. Results can be documented by photocopying the wet filter or by photography.

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3.5

DNA transfer and fixation

Transfer methods Standard protocols for gel electrophoresis, denaturation and neutralization of the gel and membranes are described in Sambrook et al. (2). Gels lacking ethidium bromide are preferred, because ethidium can cause uneven background problems. All common types of DNA transfer methods are suitable for subsequent DIG hybridization (3,4). In our experience, best results are obtained when gels are blotted by capillary transfer with 20 SSC on nylon membranes*, positively charged. Note: Alkali transfer (e.g., in 0.4 M NaOH) is not suitable for the transfer of DIG-labeled molecular weight markers*. Fixation procedure Fix the DNA to the membrane by any of the following procedures: IF you want to... UV-crosslinking (nylon membrane) THEN... Place the membrane on Whatman 3MM-paper soaked with 10 SSC. UV-crosslink the wet membrane without prior washing. After the UV-crosslinking, rinse the membrane briefly in double distilled water and allow to air-dry. Wash the membrane briefly in 2 SSC. Bake the nylon membrane at 120 C for 30 min or according to the manufacturer`s instructions. Wash the membrane briefly in 2 SSC. Bake at 80 C for 2 h under vacuum.

bake at 120 C (nylon membrane) bake at 80 C (nylon membrane) Storage of the membrane

Please refer to the following table. IF... you want to go ahead. you want to work later on THEN... Use the membrane immediately for prehybridization. store the membrane dry at 2-8 C.

3.6

Hybridization DIG Easy Hyb* ice/water shaking water-bath or hybridization oven Temperature resistant plastic or glass boxes, petri dishes, roller bottles or sealable plastic bags. Note: Do not use open containers with DIG Easy Hyb buffer.

Additional reagents and equipment required

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Hybridization temperature

The appropriate hybridization temperature is calculated according to GC content and percent homology of probe to target according to the following equation: Tm = 49.82 + 0.41 (% G + C) - (600/l) [l = length of hybrid in base pairs] Topt. = Tm 20-25 C (The given numbers of the equation were calculated according to a standard equation for hybridization solutions containing formamide, 50%.) The actual hybridization temperature Topt. for hybridization with DIG Easy Hyb is 20-25 C below the calculated Tm value. Topt. can be regarded as a stringent hybridization temperature and allows up to 18% mismatches between probe and target. When the degree of homology of your probe to template is less than 80%, you should lower Topt. accordingly (approx. 1.4C below Tm per 1 % mismatch) and also adjust the stringent washing steps accordingly (i.e. increase SSC concentration and lower washing temperature). Please refer to the following table. Step Action 1 Pre-heat an appropriate volume of DIG Easy Hyb (20 ml/100 cm2 filter) to hybridization temperature. Prehybridize filter for 30 min with gentle agitation in an appropriate container. Note: Membranes should move freely, especially if you use several membranes in the same prehybridization solution. 2 Denature DIG-labeled DNA probe (about 25 ng/ml) by boiling for 5 min and rapidly cooling in ice/water. Note: As DIG-11-dUTP is alkali-labile, DNA probes cannot be denatured by alkali treatment (NaOH). 3 Add denatured DIG-labeled DNA probe to pre-heated DIG Easy Hyb (3.5 ml/100 cm2 membrane) and mix well but avoid foaming (bubbles may lead to background). 4 Pour off prehybridization solution and add probe/hybridization mixture to membrane. Incubate at least 6 hours to O/N with gentle agitation at hybridization temperature.

Procedure

Storage of hybridization solution

DIG Easy Hyb containing DIG-labeled probe can be stored at 15 to 25 C and be reused several times when freshly denatured at 68 C for 10 min before use. Note: Do not boil DIG Easy Hyb.

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Stringency washes

For most DNA: DNA applications, a stringency wash with 0.5 SSC is sufficient. The correct post washing conditions have to be determined empirically for each probe. For human genomic DNA use 0.5 SSC and 65 C. Probes 150 bp and with a high G/C content should be washed at 68 C. For shorter probes around 100 bp or shorter, the wash temperature must be lowered. This table describes how to perform post-hybridization washes. Step 1 2 Action Wash 2 5 min in ample 2 SSC, 0.1% SDS at 15-25 C under constant agitation. Wash 2 15 min in 0.5 SSC, 0.1% SDS (prewarmed to wash temperature) at 65-68 C under constant agitation.

3.7

Immunological detection Please find in the following table composition and preparation of additional reagents required. The following buffers are also available in the DIG Wash and Block Buffer Set (Cat. No. 1 585 762) DNase and RNase free. Solution Composition / Preparation Storage and stability 15-25 C, stable 15-25 C, stable Use

Additional reagents required

Washing buffer 0.1 M Maleic acid, 0.15 M NaCl; pH 7.5 (20 C); 0.3% (v/v) Tween 20 Maleic acid 0.1 M Maleic acid, 0.15 M NaCl; buffer adjust with NaOH (solid) to pH 7.5 (20 C) Detection buffer 0.1 M Tris-HCl, 0.1 M NaCl, pH 9.5 (20 C) TE-buffer 10 mM Tris-HCl, 1 mM EDTA, pH 8.0

Washing of membrane Dilution of Blocking solution

15-25 C, Alkaline stable phosphatase buffer 15-25 C, Stopping color stable reaction

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Preparation of kit In the following table the preparation of kit working solutions is described. working solutions Solution Composition / Preparation Storage Use and stability Blocking Dissolve Blocking reagent (bottle 10) 2-8 C Preparation of stock solution 10% (w/v) in Maleic acid buffer under Blocking solution (10 conc.) constantly stirring on a heating block (65 C) or heat in a microwave oven, autoclave. The solution remains opaque. Blocking Prepare a 1 working solution by Always Blocking of solution diluting 10 Blocking solution 1:10 prepare unspecific binding with Maleic acid buffer. fresh sites Antibody Centrifuge Anti-Digoxigenin-AP (vial 8) 12 h at Binding to the solution for 5 min at 10 000 rpm in the original 2-8 C DIG-labeled probe vial prior to each use, and pipet the necessary amount carefully from the surface. Dilute Anti-Digoxigenin-AP 1:5000 (150 mU/ml) in Blocking solution. Always Visualization of ColorAdd 200 l of NBT/BCIP (vial 9) prepare antibody-binding substrate to 10 ml of Detection buffer. fresh solution Note: Store protected from light! Procedure This table describes how to perform the immunological detection on a 100 cm2 membrane. Note: All incubations should be performed at 15-25 C with agitation. If the membrane is to be reprobed, do not allow the membrane to dry at any time. Step 1 2 3 4 5 6 Action After hybridization and stringency washes, rinse membrane briefly (1-5) min in Washing buffer. Incubate for 30 min in 100 ml Blocking solution. Incubate for 30 min in 20 ml Antibody solution. Wash 2 15 min in 100 ml Washing buffer. Equilibrate 2-5 min in 20 ml Detection buffer. Incubate membrane in 10 ml freshly prepared Colorsubstrate solution in a appropriate container in the dark. Do not shake during color development. Note: The color precipitate starts to form within a few minutes and the reaction is usually complete after 16 h. The membrane can be exposed to light for short time periods to monitor color development. Stop the reaction, when desired spot or band intensities are achieved, by washing the membrane for 5 min with 50 ml of sterile double dist. water or with TE-buffer. Results can be documented by photocopying the wet filter or by photography.

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Storage of membrane

Please refer to the following table. IF... you want to reprobe the membrane THEN... the membrane should not dry off at any time, store in sealed plastic bag. Note: If you want to maintain the color, store membranes in TE buffer do not allow the membrane to dry. dry the membrane at 15-25 C for storage. Note: Color fades upon drying, to revitalize the color, wet the membrane in TE buffer.

you dont want to reprobe

3.8

Stripping and reprobing of DNA blots The alkali-labile form of DIG-11-dUTP enables easier and more efficient stripping of blots for rehybridization experiment.

General

Dimethylformamid (DMF) Additional reagents required 0.2 N NaOH, 0.1% SDS (w/v) 2 SSC Protocol Please refer to the following table. Note: When stripping and rehybridization of blots is planned, the membrane should not dry off at any time. CAUTION: Work under a fume hood Step 1 Action Heat DMF in a large glass beaker in a water bath under a fume hood to 50-60 C. Incubate the membranes in the heated DMF until the blue color precipitate is removed from the filter. CAUTION: DMF is volatile and can be ignited above 67 C. Rinse membrane briefly in double distilled water. Wash for 2 20 min in 0.2 N NaOH, SDS, 0.1% (w/v) at 37 C under constant agitation. Equilibrate briefly in 2 SSC. Air dry, or use directly for hybridization.

2 3 4 5

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4.
4.1

Results
Typical results

Genomic Southern blot

Genomic Southern Blot

Probe DIG-labeled -actin DNA fragment Template Lane 1: 100 ng DNA molecular weight marker III, digoxigenin-labeled Lane 2: 5 g human placenta DNA, Eco RI Lane 3: 2.5 g human placenta DNA, Eco RI Lane 4: 1 g human placenta DNA, Eco RI
5 2.5 1.0 g

Figure 2: This figure shows you the detection of a single-copy gene (-actin) in total human DNA using the standard protocol.

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5.
5.1

Appendix
Trouble shooting This table describes various troubleshooting parameters for DIG-labeling and detection Problem Possible cause Low sensitivity Inefficient probe labeling Recommendation Check labeling efficiency. The labeling reaction can be upscaled. Prolong incubation time to overnight. Clean up template DNA by phenolization. Use only fragments 5 kb or predigest with a restriction enzyme (e.g., four bp cutter) Make sure that template is efficiently denatured before labeling. Increase probe concentration, but use not more than 25 ng/ml DNA probe. Check hybridization and washing conditions. Prolong hybridization time. Prolong color development to 16 h. Recalculate hybridization temperature. Do not the allow the membrane to dry between prehybridization and hybridization. If you use plastic bags, remove all air bubbles prior to sealing. Some types of nylon membrane may cause high background: use nylon membrane*, especially tested for the DIG-System from Roche Diagnostics. Prolong blocking and washing steps. Check temperature of stringency washes, prewarm wash solution to correct temperature When using laboratory trays for the detection procedure, they should be rigorously cleaned before use. Anti-DIG-AP binding and color development should be done in separate trays.

Trouble shooting table

Low probe concentration in the hybridization

High background

Inefficient hybridization

Wrong type of nylon membrane

Inefficient blokking before immuno-assay Ineffective stringency washes Special hints for immuno-assay

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5.2

References

1 Hltke, H.J., Ankenbauer, W., Mhlegger, K., Rein, R., Sagner, G., Seibl, R., & Walter, T. (1995) The Digoxigenin (DIG) System for non-radioactive labeling and detection of nucleic acids-an overview. Cell. Mol. Biol. 41 (7): 883-905. 2 Sambrook, J., Fritsch, E.M. and Maniatis,T. (1989) Molecular cloning: a laboratory manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Labor, New York. 3 Southern E.M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503. 4 Khandijan, E.W. (1987) Optimized hybridization of DNA blotted and fixed to nitrocellulose and nylon membranes. Bio/Technology 5: 165.

5.3

Ordering Information

For a complete overview, please visit and bookmark our "DIG Reagents and Kits for Non-Radioactive Nucleic Acid Labeling and Detection" Special Interest Site at http://www.roche-applied-science.com/DIG

Kits

Product DIG-High Prime DNA Labeling and Detection Starter Kit I Agarose Gel DNA Extraction Kit DNA Isolation Kit for Cells and Tissue for the extraction of genomic DNA from cells and tissue ranging in size from 50 to 150 kb High Pure Plasmid Isolation Kit small scale mini-preps for sequencing, PCR, and cloning

Pack Size 1 kit ( 12 labeling reactions and 24 detection reactions) 100 reactions 10 isolations for 400 mg tissue or 5 107 cells 50 purifications 250 purifications

Cat. No. 11 745 832 910

11 696 505 001 11 814 770 001

11 754 777 001 11 754 785 001

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Single reagents

Product Blocking reagent DIG Easy Hyb (ready-to-use hybridization solution without formamide) DNA Molecular Weight Marker, Digoxigenin-labeled: DNA Molecular Weight Marker II DNA Molecular Weight Marker III DNA Molecular Weight Marker V DNA Molecular Weight Marker VI DNA Molecular Weight Marker VII DNA Molecular Weight Marker VIII DIG Wash and Block Buffer Set Hybridization bags Nylon Membrane, positively charged (20 30 cm) (10 15 cm) (0.3 3 m roll)

Pack Size 50 g 500 ml

Cat. No. 11 096 176 001 11 603 558 001

g (500 l) g (500 l) g (500 l) g (500 l) g (500 l) g (500 l) 30 blots (10 10 cm2) 50 bags

5 5 5 5 5 5

11 218 590 910 11 218 603 910 11 669 931 910 11 218 611 910 11 669 940 910 11 449 451 910 11 585 762 001 11 666 649 001

10 sheets 20 sheets 1 roll

11 209 272 001 11 209 299 001 11 417 240 001

* available from Roche Applied Science .

DISCLAIMER OF LICENSE

The labeling of nucleic acids with DIG is covered by EP patent 0 324 474 and the following US patents 5.344.757, 5.354.657 and 5.702.888 owned by Roche Diagnostics GmbH.

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