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

For general laboratory use.

Envio: shipped on gel packs

Condições de armazenamento: store at -20 °C
avoid freeze/thaw cycles

Validade: 12 months

Descrição:
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.

Contente:
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 μlTemplate requirements: oligonucleotide/ssDNA purified by HPLC or gel electrophoresis, 20 – 100 bpAdd 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. Component||Volume||Final concentration||Final molar amount PCR grade H2O||31.5 μl||n/a||n/a 5x TdT Reaction Buffer||10 μl||1x||n/a oligo-nucleotide template(1 μM)||5 μl||100 nM||5 pmol Digoxigenin-11-ddUTP (10 μM) (see 1.1)||2.5 μl||0.5 μM||50 pmol TdT (20 U/μl)||1 μl||0.4 U/μl||20 U Total volume||50 μl|||| 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/μlDegree of dioxygenylation: S1=100 %, S2=75 %, S3= 50 %, S4= 25 %, S5= 0 %Voretex and spin-down briefly. ||S1||S2||S3||S4||S5 3'-DIG oligo (500nM) (s. 3.2)||2 μl||1.5 μl||1 μl||0.5 μl||0 μl Unlab. oligo (500nM) (s. 3.1)||0 μl||0.5 μl||1 μl||1.5 μl||2 μl 1x TE Buffer pH 7.6||8 μl||8 μl||8 μl||8 μl||8 μl Total Volume||10 μl||10 μl||10 μl||10 μl||10 μl 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: ||1||2||3||4||5||6 A||10μl of S1||10μl of S2||10μl of S3||10μl of S4||10μl of S5||10μl sample B||5μl A1 + 5μl TE||5μl A2 + 5μl TE||5μl A3 + 5μl TE||5μl A4 + 5μl TE||5μl A5 + 5μl TE||5μl A6 + 5μl TE C||5μl B1 + 5μl TE||5μl B2 + 5μl TE||5μl B3 + 5μl TE||5μl B4 + 5μl TE||5μl B5 + 5μl TE||5μl B6 + 5μl TE D||5μl C1 + 5μl TE||5μl C2 + 5μl TE||5μl C3 + 5μl TE||5μl C4 + 5μl TE||5μl C5 + 5μl TE||5μl C6 + 5μl TE E||5μl D1 + 5μl TE||5μl D2 + 5μl TE||5μl D3 + 5μl TE||5μl D4 + 5μl TE||5μl D5 + 5μl TE||5μl D6 + 5μl TE F||5μl E1 + 5μl TE||5μl E2 + 5μl TE||5μl E3 + 5μl TE||5μl E4 + 5μl TE||5μl E5 + 5μl TE||5μl E6 + 5μl TE 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. 3'-OH [fmol]||100% DIG||75% DIG||50% DIG||25% DIG||0% DIG||Sample 100||2μl A1||2μl A2||...||...||...||2μl A6 50||2μl B1||...||||||||... 25||...|||||||||| 12.5|||||||||||| 6.25|||||||||||| 3.125|||||||||||| 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)."