For research use only!
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Shelf Life: 12 months
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.
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
1x TE Buffer, pH 7.6
100 ml containing 10 mM Tris-HCl, 1 mM EDTA, pH 7.6
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|
|5 μl||100 nM||5 pmol|
|Labeled UTP or ddUTP(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|
2. Estimation of Biotin 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.