Cat. No. | Amount | Price (EUR) | Buy / Note |
---|---|---|---|
APP-001 | 25 reactions x 50 μl (5 pmol each) | 286,11 | Add to Basket/Quote Add to Notepad |
For general laboratory use.
Shipping: shipped on gel packs
Storage Conditions: store at -20 °C
avoid freeze/thaw cycles
Shelf Life: 12 months
Description:
BIO 3'-End Oligonucleotide Labeling Kit with Biotin-11-UTP contains all reagents (except oligonucleotide template to be labeled and materials for Biotin detection) required for efficient 3'-End biotinylation 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 1-3 Biotin-11-UTPs to the 3'-OH group of ssDNA (e.g. an oligonucleotide) in the presence of CoCl2. It is similar to the principle of Biotin 3'-End DNA Labeling Kit (Pierce/Thermo Scientific).
The resulting 3'-End biotinylated oligonucleotides are ideally suited for applications involving sequence-specific protein binding or hybridiziation such as EMSA, Northern or Southern blots. Compared to internal, random biotinylated probes, Biotin 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
Biotin-11-UTP
5 μl, 10 mM in 10 mM Tris-HCl, pH 7.5
Unlabeled Control Oligonucleotide (60 bp)
250 μl, 1 μM in 1x TE Buffer, pH 7.6
3'-Biotin-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 Biotin-11-UTP stock solution (1 mM)
1.2 Preparation of Biotin-11-UTP working solution (10 μM)
2. 3' End Oligonucleotide labeling reaction
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 |
Biotin-11-UTP (10 μM) (see 1.2) | 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 Biotin labeling degree
Quantification of Biotin 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 Biotin moiety using streptavidin-horseradish peroxidase (HRP) or streptavidin-alkaline phosphatase (AP) conjugates.
Recommended oligonucleotide starting amount for chromogenic detection: 100 fmol
Recommended oligonucleotide starting amount for chemiluminescent detection: 20 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)
3.2 Preparation of 3'-Biotin-labeled control oligonucleotide
(3'-BIO oligo) working solution (500 nM)
3.3 Preparation of Biotin oligonucleotide standard solutions
(50 fmol/μl)
S1 | S2 | S3 | S4 | S5 | |
3'-BIO 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)
3.5 Preparation oligonucleotide standard and sample dilution series
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
3'-OH [fmol] | 100% BIO | 75% BIO | 50% BIO | 25% BIO | 0% BIO | Sample |
100 | 2μl A1 | 2μl A2 | ... | ... | ... | 2μl A6 |
50 | 2μl B1 | ... | ... | |||
25 | ... | |||||
12.5 | ||||||
6.25 | ||||||
3.125 |
3.7 Biotin detection with Streptavidin-Alkaline Phosphatase (AP)
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