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Below is a protocol for making biotinylated amplified RNA (aRNA) using one of Ambion's MEGAscript™ Kits. The reaction incorporates 2 different biotinylated nucleotides for maximum sensitivity.
Each 20 µl reaction should contain:
Assemble the reaction in the order shown. Do not assemble the reaction on ice, since spermidine in the reaction buffer can lead to precipitation of the template DNA. If multiple reactions are needed a "master mix" can be prepared by multiplying the volume of each reagent by the number of reactions.
Mix the reaction by flicking the tube and briefly microfuge to collect all of the reaction mixture at the bottom of the tube. Incubate at 37°C for 6 hrs or more (overnight incubation may increase yields). After incubation, add 1 µl DNase I (included in the MEGAscript Kit) to remove the DNA template. Incubate at 37°C for 30 min. Purify the cRNA by precipitation or by using an Ambion RNAqueous™ column. Biotinylated rNTPs can be obtained from the following suppliers:
PerkinElmer NEN
NEL542 Bio-11-CTP 10 mM, 250 nmol in 25 µl
NEL543 Bio-11-UTP 10 mM, 250 nmol in 25 µl
ENZO
42815 Bio-11-UTP 10 mM, 250 nmol in 25 µl
42818 Bio-11-CTP 10 mM, 250 nmol in 25 µl
Each 20 µl reaction should contain:
| 1.5 | µl | ds cDNA template |
| 2 | µl | 75 mM ATP |
| 2 | µl | 75 mM GTP |
| 1.5 | µl | 75 mM CTP |
| 1.5 | µl | 75 mM UTP |
| 3.75 | µl | 10 mM Bio-11-CTP |
| 3.75 | µl | 10 mM Bio-16-UTP or Bio-11-UTP |
| 2 | µl | 10X reaction buffer |
| 2 | µl | T7 RNA Polymerase |
| 20 | µl | total volume |
Assemble the reaction in the order shown. Do not assemble the reaction on ice, since spermidine in the reaction buffer can lead to precipitation of the template DNA. If multiple reactions are needed a "master mix" can be prepared by multiplying the volume of each reagent by the number of reactions.
Mix the reaction by flicking the tube and briefly microfuge to collect all of the reaction mixture at the bottom of the tube. Incubate at 37°C for 6 hrs or more (overnight incubation may increase yields). After incubation, add 1 µl DNase I (included in the MEGAscript Kit) to remove the DNA template. Incubate at 37°C for 30 min. Purify the cRNA by precipitation or by using an Ambion RNAqueous™ column. Biotinylated rNTPs can be obtained from the following suppliers:
PerkinElmer NEN
NEL542 Bio-11-CTP 10 mM, 250 nmol in 25 µl
NEL543 Bio-11-UTP 10 mM, 250 nmol in 25 µl
ENZO
42815 Bio-11-UTP 10 mM, 250 nmol in 25 µl
42818 Bio-11-CTP 10 mM, 250 nmol in 25 µl
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LT036