BISC 219/F10:RNAi General Information - Revision history

Melissa Beers at 13:55, 18 May 2011

2011-05-18T13:55:56Z

←Older revision		Revision as of 13:55, 18 May 2011
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	Perform RT-PCR (Reverse Transcriptase)to amplify the ''C. elegans'' gene of interest, using worm RNA and then cDNA as template. The RNA is isolated from treated RNAi worms and untreated worms of the same species.<BR><BR>		Perform RT-PCR (Reverse Transcriptase)to amplify the ''C. elegans'' gene of interest, using worm RNA and then cDNA as template. The RNA is isolated from treated RNAi worms and untreated worms of the same species.<BR><BR>
	Visualize the worm gene of interst in the pcr product by agarose gel electrophoresis and compare the amount of amplified gene of interest in RNAi treated vs. untreated worms.		Visualize the worm gene of interst in the pcr product by agarose gel electrophoresis and compare the amount of amplified gene of interest in RNAi treated vs. untreated worms.
-		+	<div class=noprint>
	==Links to Labs& Project Info==		==Links to Labs& Project Info==
	Series1:<BR>		Series1:<BR>
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	[[BISC 219/F10: RNAi Lab 10 \| Lab 10: Scoring your worms and RNA purification]]<br>		[[BISC 219/F10: RNAi Lab 10 \| Lab 10: Scoring your worms and RNA purification]]<br>
	[[BISC 219/F10: RNAi Lab 11 \| Lab 11: RT PCR reactions]]<br><br>		[[BISC 219/F10: RNAi Lab 11 \| Lab 11: RT PCR reactions]]<br><br>
		+	</div>

Tucker Crum: /* Outline of Experimental Design for REVERSE Genetics Project */

2010-11-29T16:27:16Z

Outline of Experimental Design for REVERSE Genetics Project

←Older revision		Revision as of 16:27, 29 November 2010
Line 41:		Line 41:
	Observe phenotype change in progeny caused by RNAi silencing or knockdown of the gene of interest compared to control worms of same strains that were NOT fed feeder strain bacteria.<BR><BR>		Observe phenotype change in progeny caused by RNAi silencing or knockdown of the gene of interest compared to control worms of same strains that were NOT fed feeder strain bacteria.<BR><BR>
	Isolate RNA from RNAi worms and control worms of same strains.<BR><BR>		Isolate RNA from RNAi worms and control worms of same strains.<BR><BR>
-	Perform RT-PCR (Reverse Transcriptase)to amplify the ''C. elegans'' gene of interest ~~and check its size~~, using worm RNA and then cDNA as template. The RNA is isolated from treated RNAi worms and untreated worms of the same species.<BR><BR>	+	Perform RT-PCR (Reverse Transcriptase)to amplify the ''C. elegans'' gene of interest, using worm RNA and then cDNA as template. The RNA is isolated from treated RNAi worms and untreated worms of the same species.<BR><BR>
	Visualize the worm gene of interst in the pcr product by agarose gel electrophoresis and compare the amount of amplified gene of interest in RNAi treated vs. untreated worms.		Visualize the worm gene of interst in the pcr product by agarose gel electrophoresis and compare the amount of amplified gene of interest in RNAi treated vs. untreated worms.

Tucker Crum: /* Outline of Experimental Design for REVERSE Genetics Project */

2010-11-24T00:59:04Z

Outline of Experimental Design for REVERSE Genetics Project

←Older revision		Revision as of 00:59, 24 November 2010
Line 42:		Line 42:
	Isolate RNA from RNAi worms and control worms of same strains.<BR><BR>		Isolate RNA from RNAi worms and control worms of same strains.<BR><BR>
	Perform RT-PCR (Reverse Transcriptase)to amplify the ''C. elegans'' gene of interest and check its size, using worm RNA and then cDNA as template. The RNA is isolated from treated RNAi worms and untreated worms of the same species.<BR><BR>		Perform RT-PCR (Reverse Transcriptase)to amplify the ''C. elegans'' gene of interest and check its size, using worm RNA and then cDNA as template. The RNA is isolated from treated RNAi worms and untreated worms of the same species.<BR><BR>
-	Visualize the worm gene of interst in the pcr product by agarose gel electrophoresis and compare ~~size~~ of amplified ~~DNA to known size of coding regions~~ gene of interest.	+	Visualize the worm gene of interst in the pcr product by agarose gel electrophoresis and compare the amount of amplified gene of interest in RNAi treated vs. untreated worms.

	==Links to Labs& Project Info==		==Links to Labs& Project Info==

Tucker Crum: /* Outline of Experimental Design for REVERSE Genetics Project */

2010-11-23T21:59:06Z

Outline of Experimental Design for REVERSE Genetics Project

←Older revision		Revision as of 21:59, 23 November 2010
Line 22:		Line 22:

	== Outline of Experimental Design for REVERSE Genetics Project ==		== Outline of Experimental Design for REVERSE Genetics Project ==
		+	'''Where are you now in this process?'''(What have you done so far; What's next?)<BR>
	Make the feeder strain of bacteria<BR>		Make the feeder strain of bacteria<BR>
	# Amplify gene of interest by pcr ;<BR>		# Amplify gene of interest by pcr ;<BR>
Line 40:		Line 41:
	Observe phenotype change in progeny caused by RNAi silencing or knockdown of the gene of interest compared to control worms of same strains that were NOT fed feeder strain bacteria.<BR><BR>		Observe phenotype change in progeny caused by RNAi silencing or knockdown of the gene of interest compared to control worms of same strains that were NOT fed feeder strain bacteria.<BR><BR>
	Isolate RNA from RNAi worms and control worms of same strains.<BR><BR>		Isolate RNA from RNAi worms and control worms of same strains.<BR><BR>
-	Perform RT-PCR (Reverse Transcriptase) ~~using the mRNA of~~ the gene of interest as template, isolated from ~~the~~ RNAi worms.<BR><BR>	+	Perform RT-PCR (Reverse Transcriptase)to amplify the ''C. elegans'' gene of interest and check its size, using worm RNA and then cDNA as template. The RNA is isolated from treated RNAi worms and untreated worms of the same species.<BR><BR>
-	Visualize ~~cDNA~~ in the pcr product by agarose gel electrophoresis and compare size of amplified ~~fragment~~ to known size of coding regions of gene of interest.	+	Visualize the worm gene of interst in the pcr product by agarose gel electrophoresis and compare size of amplified DNA to known size of coding regions gene of interest.

	==Links to Labs& Project Info==		==Links to Labs& Project Info==

Tucker Crum: /* Series 3: Reverse Genetics using RNAi in C. elegans */

2010-11-12T17:20:19Z

Series 3: Reverse Genetics using RNAi in C. elegans

←Older revision		Revision as of 17:20, 12 November 2010
Line 3:		Line 3:
	''		''
	==Series 3: Reverse Genetics using RNAi in ''C. elegans''==		==Series 3: Reverse Genetics using RNAi in ''C. elegans''==
-	In forward genetics, a mutant phenotype is attributed to one or more mutations in the DNA sequence of a gene. One disadvantage to Forward Genetics projects such as ours is that we studied a defective gene to find out how its product was functionally defective and then we had to infer the function of the normal form of the gene product. Ultimately, we care most about what that mutation can tell us about normal gene function. In our new reverse genetics projects, we will start with a normal gene rather than a mutant phenotype and we, somehow, will prevent the normal gene from producing its product and discover the function of that gene and gene product directly. In the past, gene disruption (the starting point in reverse genetics studies) of a normal gene was done in many different ways. One of the most common was by creating a "knockout": an organism in which the gene of interest was deleted, and thus "silenced", but the organism was still viable for study. Most commonly, this was done in mice. These knockout mice were VERY expensive and difficult to produce and maintain and a limited number of genes could be studied.	+	In forward genetics, a mutant phenotype is attributed to one or more mutations in the DNA sequence of a gene. One disadvantage to Forward Genetics projects, such as ours, is that we studied a defective gene to find out how its product was functionally defective and then we had to infer the function of the normal form of the gene product. Ultimately, we care most about what that mutation can tell us about normal gene function. In our new reverse genetics projects, we will start with a normal gene rather than a mutant phenotype and we, somehow, will prevent the normal gene from producing its product and discover the function of that gene and gene product directly. In the past, gene disruption (the starting point in reverse genetics studies) of a normal gene was done in many different ways. One of the most common was by creating a "knockout": an organism in which the gene of interest was deleted, and thus "silenced", but the organism was still viable for study. Most commonly, this was done in mice. These knockout mice were VERY expensive and difficult to produce and maintain and a limited number of genes could be studied.

	In recent years, the usefulness of the ''C. elegans'' model system in reverse genetic analysis of normal genes has been dramatically enhanced because this organism is particularly suited to gene silencing by RNA interference (RNAi). RNAi disrupts gene expression in an entirely different way than knocking out a gene by removing it from the genome – RNAi works by targeting specific mRNA transcripts for destruction. RNAi is a mechanism that inhibits gene function when double-stranded RNA (dsRNA) molecules that correspond to part of a “target gene” are present in a cell. By deliberately introducing defined sequences of dsRNA, biologists can observe the physiological consequences of “silencing” virtually any gene in ''C. elegans'', as well as many other plants and animals.<BR>		In recent years, the usefulness of the ''C. elegans'' model system in reverse genetic analysis of normal genes has been dramatically enhanced because this organism is particularly suited to gene silencing by RNA interference (RNAi). RNAi disrupts gene expression in an entirely different way than knocking out a gene by removing it from the genome – RNAi works by targeting specific mRNA transcripts for destruction. RNAi is a mechanism that inhibits gene function when double-stranded RNA (dsRNA) molecules that correspond to part of a “target gene” are present in a cell. By deliberately introducing defined sequences of dsRNA, biologists can observe the physiological consequences of “silencing” virtually any gene in ''C. elegans'', as well as many other plants and animals.<BR>

Tucker Crum: /* Outline of Experimental Design for REVERSE Genetics Project */

2010-11-12T16:57:43Z

Outline of Experimental Design for REVERSE Genetics Project

←Older revision		Revision as of 16:57, 12 November 2010
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	# Clean up DNA (remove enzymes); <BR>		# Clean up DNA (remove enzymes); <BR>
	# Cloning: ligate gene into vector plasmid with amp resistance gene ;<BR>		# Cloning: ligate gene into vector plasmid with amp resistance gene ;<BR>
-	# Transform competent bacterial cells ~~of a strain genetically modified to be tetracycline resistant~~;	+	# Transform competent bacterial cells;
-	# Select for transformants on media with ~~tetracycline and~~ ampicillin;<BR>	+	# Select for transformants on media with ampicillin;<BR>
	# Perform colony pcr on several transformants to be sure to find one colony containing a vector plasmid with the gene of interst		# Perform colony pcr on several transformants to be sure to find one colony containing a vector plasmid with the gene of interst
	# Culture the selected colony from colony pcr to create a lot of copies of these bacteria		# Culture the selected colony from colony pcr to create a lot of copies of these bacteria
	# Isolate the cloned plasmid DNA from that cultured colony by miniprep;<BR>		# Isolate the cloned plasmid DNA from that cultured colony by miniprep;<BR>
	# Retransform isolated plasmids (with gene interest) into HT115 (DE3)cells genetically modified to have impaired ability to degrade RNA;<BR>		# Retransform isolated plasmids (with gene interest) into HT115 (DE3)cells genetically modified to have impaired ability to degrade RNA;<BR>
-	# Select for transformants on media with ~~tetracycline and~~ ampicillin	+	# Select for transformants on media with ampicillin
	# Choose an isolated colony to culture and make lots of feeder strain bacteria; <br>		# Choose an isolated colony to culture and make lots of feeder strain bacteria; <br>
	# Induce expression of ''C. elegans'' gene dsRNA from the pL4440 vector in the bacteria by IPTG induction. <br>		# Induce expression of ''C. elegans'' gene dsRNA from the pL4440 vector in the bacteria by IPTG induction. <br>

Tucker Crum: /* Outline of Experimental Design for REVERSE Genetics Project */

2010-10-12T20:01:16Z

Outline of Experimental Design for REVERSE Genetics Project

←Older revision		Revision as of 20:01, 12 October 2010
Line 35:		Line 35:
	# Select for transformants on media with tetracycline and ampicillin		# Select for transformants on media with tetracycline and ampicillin
	# Choose an isolated colony to culture and make lots of feeder strain bacteria; <br>		# Choose an isolated colony to culture and make lots of feeder strain bacteria; <br>
		+	# Induce expression of ''C. elegans'' gene dsRNA from the pL4440 vector in the bacteria by IPTG induction. <br>
	# Seed NM lite worm growth media plates with feeder strain produced as described <BR>		# Seed NM lite worm growth media plates with feeder strain produced as described <BR>
	Plate wild type ''C. elegans'' worms (N2 and rrf-3 strains) on feeder plates made as described (containing bacteria expressing dsRNA of our gene of interest). <BR><BR>		Plate wild type ''C. elegans'' worms (N2 and rrf-3 strains) on feeder plates made as described (containing bacteria expressing dsRNA of our gene of interest). <BR><BR>

Tucker Crum: /* Outline of Experimental Design for REVERSE Genetics Project */

2010-10-12T15:35:40Z

Outline of Experimental Design for REVERSE Genetics Project

←Older revision		Revision as of 15:35, 12 October 2010
Line 37:		Line 37:
	# Seed NM lite worm growth media plates with feeder strain produced as described <BR>		# Seed NM lite worm growth media plates with feeder strain produced as described <BR>
	Plate wild type ''C. elegans'' worms (N2 and rrf-3 strains) on feeder plates made as described (containing bacteria expressing dsRNA of our gene of interest). <BR><BR>		Plate wild type ''C. elegans'' worms (N2 and rrf-3 strains) on feeder plates made as described (containing bacteria expressing dsRNA of our gene of interest). <BR><BR>
-	Observe phenotype change in progeny caused by RNAi silencing or knockdown of the gene of interest compared to control worms of same strains that ~~we did~~ NOT fed feeder strain bacteria.<BR><BR>	+	Observe phenotype change in progeny caused by RNAi silencing or knockdown of the gene of interest compared to control worms of same strains that were NOT fed feeder strain bacteria.<BR><BR>
	Isolate RNA from RNAi worms and control worms of same strains.<BR><BR>		Isolate RNA from RNAi worms and control worms of same strains.<BR><BR>
	Perform RT-PCR (Reverse Transcriptase) using the mRNA of the gene of interest as template, isolated from the RNAi worms.<BR><BR>		Perform RT-PCR (Reverse Transcriptase) using the mRNA of the gene of interest as template, isolated from the RNAi worms.<BR><BR>

Tucker Crum: /* Outline of Experimental Design for REVERSE Genetics Project */

2010-10-12T15:34:09Z

Outline of Experimental Design for REVERSE Genetics Project

←Older revision		Revision as of 15:34, 12 October 2010
Line 37:		Line 37:
	# Seed NM lite worm growth media plates with feeder strain produced as described <BR>		# Seed NM lite worm growth media plates with feeder strain produced as described <BR>
	Plate wild type ''C. elegans'' worms (N2 and rrf-3 strains) on feeder plates made as described (containing bacteria expressing dsRNA of our gene of interest). <BR><BR>		Plate wild type ''C. elegans'' worms (N2 and rrf-3 strains) on feeder plates made as described (containing bacteria expressing dsRNA of our gene of interest). <BR><BR>
-	Observe phenotype change in progeny caused by RNAi silencing or knockdown of the gene of interest compared to control worms of same strains that we NOT fed feeder strain bacteria.<BR><BR>	+	Observe phenotype change in progeny caused by RNAi silencing or knockdown of the gene of interest compared to control worms of same strains that we did NOT fed feeder strain bacteria.<BR><BR>
	Isolate RNA from RNAi worms and control worms of same strains.<BR><BR>		Isolate RNA from RNAi worms and control worms of same strains.<BR><BR>
	Perform RT-PCR (Reverse Transcriptase) using the mRNA of the gene of interest as template, isolated from the RNAi worms.<BR><BR>		Perform RT-PCR (Reverse Transcriptase) using the mRNA of the gene of interest as template, isolated from the RNAi worms.<BR><BR>

Tucker Crum: /* Outline of Experimental Design for REVERSE Genetics Project */

2010-10-12T15:15:35Z

Outline of Experimental Design for REVERSE Genetics Project

←Older revision		Revision as of 15:15, 12 October 2010
Line 22:		Line 22:

	== Outline of Experimental Design for REVERSE Genetics Project ==		== Outline of Experimental Design for REVERSE Genetics Project ==
-	~~'''Where are you now in this process?'''(What have you done so far; What's next?)<BR>~~
	Make the feeder strain of bacteria<BR>		Make the feeder strain of bacteria<BR>
	# Amplify gene of interest by pcr ;<BR>		# Amplify gene of interest by pcr ;<BR>