3'-End Oligonucleotide Labeling Reagent Kit

For general laboratory use.

Envio: shipped on gel packs

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

Validade: 12 months

Descrição:
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.

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

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 reactionStore 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
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.