Cat. No. | Amount | Price (EUR) | Buy / Note |
---|---|---|---|
APP-003 | 25 reactions x 50 μl (5 pmol each) | 234,09 | 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:
3'-End Oligonucleotide Labeling Reagent Kit contains all buffer reagents required for efficient 3'-End Labeling of DNA oligonucleotides (length: 20 -100 bp, 5 pmol per reaction) except of oligonucleotide template to be labeled and labeled nucleotides..
The labeling principle is based on Terminal deoxynucleotidyl Transferase (TdT) that template-independently transfers labeled nucleotides to the 3'-OH group of ssDNA (e.g. an oligonucleotide) in the presence of CoCl2. The number of nucleotide and thus label incorporation depends on the type of nucleotide (UTP/ddUTP) and type of label.
Labeled UTP: 1 – 3 label (average)
Labeled dUTP: multiple label (tail length is highly nucleotide specific)
Labeled ddUTPs: 1 label
The resulting 3'-End labeled oligonucleotides are ideally suited for applications involving sequence-specific protein binding or hybridiziation such as EMSA, Northern or Southern blots. Compared to internal, random labeled probes, the label 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
Unlabeled Control Oligonucleotide (60 bp)
250 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. 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 |
Labeled UTP or ddUTP(10 μM) | 2.5 μl | 0.5 μM | 50 pmol |
TdT (20 U/μl) | 1 μl | 0.4 U/μl | 20 U |
Total volume | 50 μl |
2. Estimation of labeling degree
Quantification of labeling degree is essential for reproducible downstream results.
Biotin or Digoxigenin-labeled oligonucleotides can be indirectly detected via Streptavidin or anti-Digoxigenin conjugates, respectively.
The labeling degree of fluorescent oligonucleotides can be directly detected by measurement of the nucleic acid-dye conjugate absorbance followed by a calculation of dye to base ratio according to the law of Lambert-Beer.
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