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BISC219/F11: RNAi Lab 9: Difference between revisions
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== Assignment == | == Assignment == | ||
Remember to check the Assignment section of the wiki for instructions about the graded assignment due in the next lab and check the Weekly Calendar for other work to accomplish before the next lab. | Remember to check the Assignment section of the wiki for instructions about the graded assignment due in the next lab and check the Weekly Calendar for other work to accomplish before the next lab. | ||
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== 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> | |||
'''A.''' Make the feeder strain of bacteria<BR> | |||
# Amplify gene of interest by PCR <BR> | |||
# Restriction Enzyme digestion of amplified DNA to create "sticky ends" for ligation<BR> | |||
# Clean up DNA (remove enzymes) <BR> | |||
# Cloning: ligate gene into vector plasmid with amp resistance gene <BR> | |||
# Transform competent bacterial cells | |||
# 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 interest | |||
# 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> | |||
# 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 | |||
# 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> | |||
'''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> | |||
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