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BISC219/F11: RNAi General Information: Difference between revisions
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→Outline of Experimental Design for REVERSE Genetics Project
(New page: {{Template:BISC 219/F10}} <div style="padding: 10px; width: 720px; border: 5px solid #2171B7;"> '' ==Series 3: Reverse Genetics using RNAi in ''C. elegans''== In forward genetics, a mutant...) |
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== 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 | '''Where are you now in this process?'''(What have you done so far? What's next?)<BR> | ||
Make the feeder strain of bacteria<BR> | A. Make the feeder strain of bacteria<BR> | ||
# Amplify gene of interest by PCR | # Amplify gene of interest by PCR <BR> | ||
# Restriction Enzyme digestion of amplified DNA to create "sticky ends" for ligation | # Restriction Enzyme digestion of amplified DNA to create "sticky ends" for ligation<BR> | ||
# Clean up DNA (remove enzymes) | # Clean up DNA (remove enzymes) <BR> | ||
# Cloning: ligate gene into vector plasmid with amp resistance gene | # Cloning: ligate gene into vector plasmid with amp resistance gene <BR> | ||
# Transform competent bacterial cells | # Transform competent bacterial cells | ||
# Select for transformants on media with ampicillin | # 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 | # Perform colony pcr on several transformants to be sure to find one colony containing a vector plasmid with the gene of interest | ||
# 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 | # 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 | # 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 ampicillin | # Select for transformants on media with ampicillin | ||
# Choose an isolated colony to culture and make lots of feeder strain bacteria | # 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 | # 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> | ||
B. 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> | |||
C. 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> | |||
