DIG 3'-End Oligonucleotide Labeling Kit with Digoxigenin-11-ddUTP

For general laboratory use.

Shipping: shipped on gel packs

Storage Conditions: store at -20 °C
avoid freeze/thaw cycles

Shelf Life: 12 months

Description:
DIG 3'-End Oligonucleotide Labeling Kit with Digoxigenin-11-ddUTP contains all reagents (except oligonucleotide template to be labeled and materials for Digoxigenin detection) required for efficient 3'-End digoxigenylation of DNA oligonucleotides (length: 20 -100 bp, 5 pmol per reaction).

The labeling principle is based on Terminal deoxynucleotidyl Transferase (TdT) that template-independently transfers 1 Digoxigenin-11-ddUTP to the 3'-OH group of ssDNA (e.g. an oligonucleotide) in the presence of CoCl₂. It is similar to the principle of DIG Oligonucleotide 3'-End DNA Labeling Kit, 2nd Generation (Roche).

The resulting 3'-End digoxigenylated oligonucleotides are ideally suited for applications involving sequence-specific protein binding or hybridiziation such as EMSA, Northern or Southern blots. Compared to internal, random digoxigenylated probes, Digoxigenin is located at the 3'-End only and less likely interferes with probe binding.

TdT possesses a preference for single-stranded DNA (ssDNA) over dsDNA with 3'-overhangs or blunt ends. For the preparation of labeled dsDNA complexes, label each complementary oligonucleotide separately and anneal them before use.

Content:
Terminal Deoxynucleotidyl Transferase (TdT)
30 μl (20 U/μl) in 100 mM potassium acetate (pH 6.8), 2 mM 2-mercaptoethanol, 0.01% Triton X-100 (v/v) and 50% glycerol (v/v)

5x TdT Reaction Buffer
400 μl containing 1 M potassium cacodylate, 0.125 M Tris, 0.05% Triton X-100 (v/v), 5 mM CoCl₂, pH 7.2

Digoxigenin-11-ddUTP
25 μl (1 mM )

Unlabeled Control Oligonucleotide (60 bp)
250 μl, 1 μM in 1x TE Buffer, pH 7.6

3'-Digoxigenin-labeled Control Oligonucleotide (60 bp)
130 μl, 1 μM in 1x TE Buffer, pH 7.6

PCR-grade H₂O
12.5 ml

1x TE Buffer, pH 7.6
100 ml containing 10 mM Tris-HCl, 1 mM EDTA, pH 7.6

Stop Buffer
400 μl, 0.5 M EDTA solution, pH 8

1. Preparation of working solutions

1.1 Preparation of Digoxigenin-11-ddUTP working solution (10 μM)

• Thaw 1 mM Digoxigenin-11-ddUTP solution on ice, voretex and spin-down briefly.
• Prepare a 1: 100 dilution with PCR-grade H2O to achieve a final concentration of 10 μM (e.g. 1 μl of 1 mM Digoxigenin-11-ddUTP + 99 μl PCR-grade H2O).
• Keep working solution (10 μM) on ice until use (see 2.).
• Prepare Digoxigenin-11-ddUTP working solution (10 μM) freshly for each experiment. Do not store for subsequent use.

2. 3' End Oligonucleotide labeling reaction

• Store all components except of TdT on ice until use.
• Store TdT at -20°C until use.
• Final Assay volume: 50 μl
• Template requirements: oligonucleotide/ssDNA purified by HPLC or gel electrophoresis, 20 – 100 bp
• Add all components on ice exactly in the order listed below.
• Mix reaction gently by pipetting up and down. Do not voretex!
• Incubate 30 min at 37 °C.
• Add 1 μl Stop Buffer (0.5 M EDTA solution, pH 8) to stop each reaction.
• Store reactions on ice for subsequent use (see 3.) or -20 °C for long-term storage.

3. Estimation of Digoxigenin labeling degree
Quantification of Digoxigenin labeling degree is essential for reproducible downstream results. An oligonucleotide dilution series is immobilized on a positively-charged membrane (Dot Blot) followed by an indirect detection of the Digoxigenin moiety using an anti-Digoxigenin-alkaline phosphatase (AP) conjugate.

Recommended oligonucleotide starting amount for chemiluminescent detection: 100 fmol
The following reagent amounts (3.1 – 3.5) are calculated for an oligonucleotide starting amount of 100 fmol.

3.1 Preparation of Unlabeled control oligonucleotide (Unlab. oligo)
working solution (500 nM)

• Thaw 1 μM unlabeled control oligonucleotide solution on ice, voretex and spin-down briefly.
• Prepare a 1:2 dilution with 1x TE Buffer, pH 7.6 to achieve a final concentration of 500 nM (e.g. 5 μl of 1 μM unlabeled control oligonucleotide + 5 μl 1x TE Buffer, pH 7.6).
• Keep the working solution (500 nM) on ice until use (see 2.).
• Prepare unlabeled control oligonucleotide working solution (500 nM) freshly for each experiment. Do not store for subsequent use.

3.2 Preparation of 3'-Digoxigenin-labeled control oligonucleotide
(3'-DIG oligo) working solution (500 nM)

• Thaw 1 μM 3'-Digoxigenin-labeled control oligonucleotide solution on ice, voretex and spin-down briefly.
• Prepare a 1:2 dilution with 1x TE Buffer,pH 7.6 to achieve a final concentration of 500 nM (e.g. 5 μl of 1 μM 3'-Digoxigenin-labeled control oligonucleotide solution + 5 μl 1x TE Buffer, pH 7.6).
• Keep the working solution (500 nM) on ice until use (see 2.)
• Prepare 3'-Digoxigenin-labeled control oligonucleotide working solution (500 nM) freshly for each experiment. Do not store for subsequent use.

3.3 Preparation of Digoxigenin oligonucleotide standard solutions
(50 fmol/μl)

• Prepare Digoxigenin oligonucleotide standard solutions (S1 – S5) with varying degrees of oligonucleotide digoxigenylation as follows.
• Total oligonucleotide concentration (S1-S5): 50 fmol/μl
• Degree of dioxygenylation: S1=100 %, S2=75 %, S3= 50 %, S4= 25 %, S5= 0 %
• Voretex and spin-down briefly.

3.4 Preparation of sample dilutions (50 fmol/μl)

• Dilute sample labeling reaction(s) (see 1) 1:2 to a final oligonucleotide concentration of 50 nM (e.g. 5 μl sample labeling reaction + 5 μl 1x TE Buffer, pH 7.6.)
• Voretex and spin-down briefly.

3.5 Preparation oligonucleotide standard and sample dilution series

• Transfer 10 μl of each Digoxigenin oligonucleotide standard solution S1 – S5 (see 2.1) to well A1 – A5 of a low absorption 96-well PCR plate, respectively (e.g. 96-well Multiply® PCR plate, Sarstedt, #72.1979.102).
• Transfer 10 μl of sample dilution(s) (see 2.2) to the remaining A" wells (A6 to A…)
• Prepare a two-fold dilution series with 1x TE Buffer as follows:

3.6 Dot Blot and UV crosslinking

• Equilibrate a positively-charged membrane of appropriate size for at least 10 minutes in 1x TE Buffer, pH 7.6 (e.g. Biorad Zeta-Probe® Membrane, #1620159).
• Place the equilibrated membrane onto a clean dry Whatman® paper. Allow excess buffer to absorb into the membrane, but do not let the membrane dry out.
• Spot 2 μl of each dilution onto the membrane.

• Allow samples to absorb into the membrane.
• Immediately fix the oligonucleotide to the membrane by crosslinking with UV-light using a commercial UV-light crosslinking instrument according to the manufacturers instructions (e.g. 120 mJ/cm², 254 nm bulbs, 45-60 second exposure).
• Proceed immediately with detection (see 3.7) or store the membrane dry at room-temperature.

3.7 Digoxigenin detection with anti-Digoxigenin-Alkaline Phosphatase (AP)

• Perform Digoxigenin detection with an appropriate anti-Digoxigenin-alkaline phosphatase (AP) conjugate followed by chemiluminescent.
• Compare spot intensities of sample lanes to those of control oligonucleotide template and Digoxigenin oligonucleotide standard.
  Please note: Digoxigenin labeling degree may vary depending on the template (e.g. purity, length or overall sequence).

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