Q-PCR with Strand Curve

DNase Treatment: with Ambion DNaseI DNAfree kit

Incubate at 37°C 30 min, add 1μl rDNaseI, and incubate 37°C 1 hr. Add 10μl DNase Inactivation Reagent, incubate 2 min at room temp, centrifuge 10,000xg 1.5 min, and transfer 50μl to new tube. Precipitate with 5μl 3M NaOAc + 1μl glycogen + 150μl EtOH in -20°C overnight. Spin down RNA at 13,000rpm 30 min, remove sup, dry pellet 10 min, and resuspend pellet in 20μl DEPC H2O. Spec RNA.

Reverse Transcription Reaction: with Applied Biosystems Taqman kit

For Bulk RT samples used for standard curves, use wt RNA (i.e. Eatro, 29-13, ect.) or - tet RNA, and make a - RT sample with +RT sample. Incubate at 25°C for 10 min, 48°C for 45 min, 95°C for 5 min, cool on ice, then add 1.5μl RNaseH (Ambion/Applied Biosystems), incubate at 37°C for 20 min, then at 65°C for 20 min.

Q-PCR Setup

Based on RNA used for cDNA, cDNA = 80ng/μL.

Following plate map (example in Q-PCR plate map file) add 2.5μL of cDNA to each well. Mix 1.5μM primers with SYBR green Master Mix in a tube (10μl primers to 12.5μl SYBR green per well for as many wells as needed) and add 22.5μL of primer/SYBR mix to each well. Cover plate with adhesive cover, use squeegee to press down cover, and place in machine.

Use program:

Download Protocol: Q-PCR Protocol, Q-PCR Plate Map
(note requires Microsoft Word to open)