Designing and Preparing dsRNA: Difference between revisions

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__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

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==Preparing dsRNA==

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

===Template Preparation===

*set up a 250 uL PCR reaction

**50 uL 10X KOD ~~byffer~~

**25 uL 10X KOD buffer

**50 uL ~~dNTPs~~

**25 uL dNTPs

**30 uL MgCl (this can be adjusted to optimize PCR conditions)

**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

**Water to bring up to 250 uL

**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).

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*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.

*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

@@ Line 1: / Line 1: @@
+__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
@@ Line 5: / Line 9: @@
 ==Preparing dsRNA==
-*see http://www.flyrnai.org/DRSC-PRS.html
 ===Template Preparation===
 *set up a 250 uL PCR reaction
-**50 uL 10X KOD byffer
+**25 uL 10X KOD buffer
-**50 uL dNTPs
+**25 uL dNTPs
-**30 uL MgCl (this can be adjusted to optimize PCR conditions)
+**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
-**Water to bring up to 250 uL
+**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).
@@ Line 35: / Line 38: @@
 *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.
+*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