Difference between revisions of "Designing and Preparing dsRNA"
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| + | __NOTOC__ | ||
| + | [[Category:knockdown]] | ||
| + | [[Category:Cell Culture]] | ||
| + | [[Category:Drosophila]] | ||
==Ordering dsRNA Primers== | ==Ordering dsRNA Primers== | ||
*If available, choose primers from Harvard DSRC site http://www.flyrnai.org/cgi-bin/RNAi_gene_lookup_public.pl | *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''') | *Add T7 RNA Polymerase sites to the 5' of each primer ('''TAATACGACTCACTATAGG''') | ||
*Record primer and amplicon (DSRC) | *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. | ||
| + | *Label RNA with concentration and store at -20 | ||
| + | |||
| + | *see PMID 18388942, PMID 18265350 , PMID 11752672 and http://www.flyrnai.org/DRSC-PRS.html | ||
Latest revision as of 14:31, 1 October 2009
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
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.
- Label RNA with concentration and store at -20
