Add T7 RNA Polymerase sites to the 5' of each primer (TAATACGACTCACTATAGG)
Record primer and amplicon (DSRC)
Preparing dsRNA
Template Preparation
set up a 250 uL PCR reaction
25 uL 10X KOD buffer
25 uL dNTPs
50 uL Primers (sense and antisense combined and diluted to 1 uM. 2 uL of each primer plus 196 uL of water)
15 uL MgCl (this can be adjusted to optimize PCR conditions)
Template (previously prepared PCR product or 1 uL of cDNA)
2 uL KOD polymerase
132uL Water (bring up to 250 uL)
Run using TD-KOD program
transfer PCR product to a clean tube. Add 25 uL of 3M NaAcetate (pH 5.2) and 500 uL ice cold ethanol (stored at -20).
Incubate on ice for >20 min.
Centrifuge 5 min at 4C on high in eppendorf centrifuge.
Carefully aspirate supernatant. Add 1 mL 70% ethanol and centrifuge again.
Aspirate supernatant and let pellet air dry.
Resuspend in 20 uL EB and measure concentration by nanodrop.
RNA Synthesis
Prepare RNA synthesis reaction (RiboMAX Large Scale RNA Preparation Kit):
20 uL T7 Buffer
30 uL rNTPs (prepare by combining equal volmes of rNTP together)
10 uL Enzyme mix
10 ug PCR product
water to bring volume up to 100 uL
Place in PCR machine and incubate overnight at 37C (37 hold program)
Quantify RNA
Dilute RNA 10x in loading dye
Load 1 and 3 uL of diluted RNA, plus 4 or 8 uL of 1 kB Plus DNA Ladder
Using ImageJ, quantify ladder and dsRNA bands and calculate concentration. See Using ImageJ to Quantify Bands. Check that the dilutions are close to 3x apart in signal intensity.