View source for Designing and Preparing dsRNA

__NOTOC__
[[Category:knockdown]]
[[Category:Cell Culture]]
[[Category:Drosophila]]
==Ordering dsRNA Primers==
*If available, choose primers from Harvard DSRC site http://www.flyrnai.org/cgi-bin/RNAi_gene_lookup_public.pl
*Add T7 RNA Polymerase sites to the 5' of each primer ('''TAATACGACTCACTATAGG''')
*Record primer and amplicon (DSRC)

==Preparing dsRNA==
*see http://www.flyrnai.org/DRSC-PRS.html

===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.  Check that the dilutions are close to 3x apart in signal intensity.
*Label RNA with concentration and store at -20