Peter Hauser (last update 3.9.2002)

using RiboAMP™ RNA Amplification Kit

http://www.arctur.com/images/pdf/riboamp_user_guide.pdf

Procedure

1st strand synthesis:

• Prepare RNA sample (about 1 µg) in 10µl nuclease free water
• Add 1µl Primer A, mix and spin down
• Incubate at 65°C for 5 minutes, chill to 4°C
• Add 7µl 1st Strand Master Mix and 2µl 1st Strand Enzyme Mix to the sample
•  Incubate at 42°C for 45 minutes
•  Add 2µl 1st Strand Nuclease Mix, mix and incubate at 37°C for 20 minutes
• To inactivate nuclease, incubate for 5 minutes at 95°C

2nd strand synthesis:

• Add 1µl Primer B to the 1st strand mix
• Incubate 2 minutes at 95°C, then chill to 4°C
• For the reaction mix add 29µl 2nd Strand Master Mix and 1µl 2nd Strand Enzyme Mix to the chilled sample
• Incubate sample: 5 minutes at 25°C, 10 minutes at 37°C, 5 minutes at 70°C. Keep on 4°C till purification (max. 30 minutes)

cDNA purification:

• Equilibrate purification column with 250 µl DNA binding buffer DB, centrifuge at 16.000g for 1 minute
• Add 200µl binding buffer DB to sample and apply to purification column
• Centrifuge at 100g for 2 minutes, followed by 1 minute at 10.000g
• Wash by adding 250µl DNA wash buffer DW and centrifuge 2 minutes at 16.000g
• Place column in a new collection tube and add 16 µl DNA elution buffer DE
• Incubate 1 minute at room temperature
• Elute by centrifugation at 1000g for 1 minute, followed by 1 minute at 16.000g

In Vitro Transcription:

• To the 16 µl cDNA add: 8µl IVT Buffer, 12µl IVT Master Mix and 4 µl Enzyme Mix
• Incubate the reaction mix for 4 hours at 42°C
• Chill the samples to 4°C, then add 2µl DNase Mix and incubate at 37°C for 15 minutes

aRNA purification:

• Equilibrate purification column with 250 µl RNA binding buffer RB, centrifuge at 16.000g for 1 minute
• Add 200µl RNA binding buffer RB to sample and apply to purification column
• Centrifuge at 100g for 2 minutes, followed by 1 minute at 10.000g
• Wash by adding 200µl RNA wash buffer RW and centrifuge 1 minute at 10.000g
• Wash a 2nd time by adding 250µl RNA wash buffer RW, centrifuge 2 minutes at 16.000g
• Place column into a new tube and apply 30µl RNA elution buffer RE on to column, incubate 1 minute at room temperature
• Elute by centrifugation with 1 minute at 1000g, followed by 1 minute at 16.000g

For all thermal reactions use a PCR thermo cycler with heated lid.
Store aRNA at -70°C.

Material:

RiboAmp RNA Amplification Kit  |  Arcturus, Mountain View; CA #KIT0201