Bitan:biotinylation(oligo)

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

=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 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