Designing and Preparing dsRNA - Revision history

Davebrid at 14:31, 1 October 2009

2009-10-01T14:31:50Z

← Older revision		Revision as of 14:31, 1 October 2009
Line 38:		Line 38:
	*Dilute RNA 10x in loading dye		*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		*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		*Label RNA with concentration and store at -20

	*see PMID 18388942, PMID 18265350 , PMID 11752672 and http://www.flyrnai.org/DRSC-PRS.html		*see PMID 18388942, PMID 18265350 , PMID 11752672 and http://www.flyrnai.org/DRSC-PRS.html

Davebrid: /* Preparing dsRNA */

2009-09-16T15:40:37Z

Preparing dsRNA

← Older revision		Revision as of 15:40, 16 September 2009
Line 9:		Line 9:

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

	===Template Preparation===		===Template Preparation===
	*set up a 250 uL PCR reaction		*set up a 250 uL PCR reaction
Line 42:		Line 40:
	*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. Check that the dilutions are close to 3x apart in signal intensity.
	*Label RNA with concentration and store at -20		*Label RNA with concentration and store at -20

			*see PMID 18388942, PMID 18265350 , PMID 11752672 and http://www.flyrnai.org/DRSC-PRS.html

Davebrid at 15:38, 16 September 2009

2009-09-16T15:38:07Z

← Older revision		Revision as of 15:38, 16 September 2009
Line 19:		Line 19:
	**Template (previously prepared PCR product or 1 uL of cDNA)		**Template (previously prepared PCR product or 1 uL of cDNA)
	**2 uL KOD polymerase		**2 uL KOD polymerase
	**Water to bring up to 250 uL		**132uL Water (bring up to 250 uL)
	**Run using TD-KOD program		**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).		*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).

Davebrid at 19:36, 14 September 2009

2009-09-14T19:36:58Z

← Older revision		Revision as of 19:36, 14 September 2009
Line 13:		Line 13:
	===Template Preparation===		===Template Preparation===
	*set up a 250 uL PCR reaction		*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)		**Template (previously prepared PCR product or 1 uL of cDNA)
	**2 uL KOD polymerase		**2 uL KOD polymerase

Davebrid at 19:34, 14 September 2009

2009-09-14T19:34:20Z

← Older revision		Revision as of 19:34, 14 September 2009
Line 1:		Line 1:
			__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

Davebrid: added details about dsRNA preparation

2009-09-14T19:33:43Z

added details about dsRNA preparation

← Older revision		Revision as of 19:33, 14 September 2009
Line 6:		Line 6:
	==Preparing dsRNA==		==Preparing dsRNA==
	*see http://www.flyrnai.org/DRSC-PRS.html		*see http://www.flyrnai.org/DRSC-PRS.html

			===Template Preparation===
			*set up a 250 uL PCR reaction
			**50 uL 10X KOD byffer
			**50 uL dNTPs
			**30 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
			**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

Davebrid at 13:47, 3 August 2009

2009-08-03T13:47:43Z

← Older revision		Revision as of 13:47, 3 August 2009
Line 3:		Line 3:
	*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==
			*see http://www.flyrnai.org/DRSC-PRS.html

Davebrid at 13:44, 3 August 2009

2009-08-03T13:44:42Z

← Older revision		Revision as of 13:44, 3 August 2009
Line 1:		Line 1:
	==Ordering dsRNA Primers==		==Ordering dsRNA Primers==
	*If available choose primers from Harvard DSRC site http://www.flyrnai.org/		*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:		*Add T7 RNA Polymerase sites to the 5' of each primer ('''TAATACGACTCACTATAGG''')
	'''TAATACGACTCACTATAGG'''		*Record primer and amplicon (DSRC)

Davebrid: intial page

2009-08-03T13:27:39Z

intial page

New page

==Ordering dsRNA Primers==
*If available choose primers from Harvard DSRC site http://www.flyrnai.org/
*Add T7 RNA Polymerase sites to the 5' of each primer:
'''TAATACGACTCACTATAGG'''