Bitan:biotinylation(oligo)

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For an ELONA experiment you may need to biotinylate your aptamer/oligonucleotide. This protocol starts with dsDNA oligo and ends with biotinylated ssRNA.

Contents

DNA aptamer PCR

100 ul reaction: 10 mM dNTPs 10 ul
10x buffer 10 ul
Forward Primer 1 ul
Reverse Primer 1 ul
Template DNA 3 ul
H2O 64 ul
Taq 1 ul
Program: "Aptamar2" (Kazuma)

Aptamer Transcription

100 ul reaction:
100 mM rNTPs 7.5 ul ea.
(ATP, CTP, GTP + UTP)
5x Buffer 20 ul
Template DNA 40 ul
T7 Enzyme Mix 10 ul
Incubate 4 hours at 37oC
Optional control: 20 ul reaction with luciferase template

DNA digestion

Add 10 ul DNase 1 to 100 ul transcription reaction
Incubate 30 min. at RT

Chloroform Preparation

1 To the reaction volume add an equal volume of Phenol:Chloroform:Isoamyl alcohol (125:24:1)
2 Mix thoroughly
3 Centrifuge at top speed for 2 min.
4 Remove and discard the bottom layer
Repeat 1-4 with Chloroform:Isoamyl Alcohol (24:1)

Ethanol Precipitation

Add 0.1 volumes 3 M Sodium Acetate
Add 3-4 volumes 100% EtOH
Mix quickly
Incubate at -20oC for at least 10 min
Centrifuge at 4oC for at least 20 min (program 8)
check tubes for radiation contamination
Pour off or aspirate supernatant and wash pellet with 1 ml 70% EtOH
Mix well and centrifuge 10 min
Pour off EtOH supernatant and let residual EtOH evaporate at 37oC
Suspend RNA sample in TE buffer or Nuclease-Free H2O (same volume as original reaction)
Incubate at 65oC to resuspend
Store at -20oC

Biotinylation

There is an oligonucleotide biotinylation kit in the hallway freezer, it has the necessary buffers and reagents.
Mix in a 1.5 ml centrifuge tube on ice:
100 pmol oligonucleotide

5x TdT Buffer 4 ul
Template X ul
25 mM CoCl2 4 ul
1 mM Biotin-ddUTP1 ul
Terminal Transferase1 ul
ddH2OY ul
Final Volume 20 ul

Mix the reaction thoroughly and centrifuge briefly
Incubate 1 h at 37 oC
Stop the reaction by adding 2 ul of 0.2 M EDTA

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