|
|
| Line 2: |
Line 2: |
| <div style="padding: 10px; width: 720px; border: 5px solid #7E354D;"> | | <div style="padding: 10px; width: 720px; border: 5px solid #7E354D;"> |
|
| |
|
| ==Series 3 Reverse Genetics: Scoring RNAi Worms == | | ==Series 3 Investigation of Gene Regulation Using RNAi-- == |
| Today in lab you will examine your RNAi worms and their progeny. <br>
| | Placeholder for future information |
| #Examine worms with a known bli-1 mutation to see what knockdown looks like.
| |
| #Compare your RNAi worms to worms that have a known ''bli-1'' mutation - how do they compare?
| |
| #Examine your control worms to review wild type phenotype in comparison to mutants
| |
| #Compare your RNAi worms to the control worms - are they the same phenotype? Different? What do the “control” worms tell you?
| |
| #Examine your RNAi’d wild type (N2) and then your RNAi’d ''rrf-3'' worms. Do you see any differences between the two treated strains of worms?
| |
|
| |
|
| <br>
| |
| Be sure to record all of your results in your lab notebook.<br>
| |
| Take pictures of your control and RNAi worms to use in the results section of your next paper.<br>
| |
| <br>
| |
| == 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.
| |
| <div class=noprint>
| |
| == 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>
| |
| </div> | | </div> |
| <div class=noprint> | | <div class=noprint> |
| Line 53: |
Line 22: |
| '''Series3:'''<BR> | | '''Series3:'''<BR> |
| [[BISC219/F12:RNA interference | RNA interference]]<BR> | | [[BISC219/F12:RNA interference | RNA interference]]<BR> |
| [[BISC219/F12: RNAi General Information | RNAi General Information]] <br> | | [[BISC219/F12: RNAi General Information | Background Info on Project 3]] <br> |
| [[BISC219/F12: Media Recipes | Media Recipes]]<br> | | [[BISC219/F12: Media Recipes | Media Recipes]]<br> |
| [[BISC219/F12: RNAi Lab 5 | Lab 5: Picking your gene to RNAi]]<br>
| | [[BISC219/F12: RNAi Lab 7 | Lab 7: ]]<br> |
| [[BISC219/F12: RNAi Lab 6 | Lab 6: Cloning your gene of interest]]<br>
| | [[BISC219/F12: RNAi Lab 8 | Lab 8: ]]<br> |
| [[BISC219/F12: RNAi Lab 7 | Lab 7: Picking your transformant]]<br> | | [[BISC219/F12: RNAi Lab 9 | Lab 9: i]]<br> |
| [[BISC219/F12: RNAi Lab 8 | Lab 8: Plasmid purification and transformation]]<br> | | [[BISC219/F12: RNAi Lab 10 | Lab 10: ]]<br> |
| [[BISC219/F12: RNAi Lab 9 | Lab 9: Induction of bacteria for RNAi]]<br> | |
| [[BISC219/F12: RNAi Lab 10 | Lab 10: Scoring your worms]]<br> | |
| [[BISC219/F12: RNAi Lab 11 | Lab 11: ]]<br><br> | | [[BISC219/F12: RNAi Lab 11 | Lab 11: ]]<br><br> |
| </div> | | </div> |
