||Outside of Lab Work
||Aug 30 to
|LEARN WORM HUSBANDRY;
View worm videos;
Make your worm pick;
Examine worms: recognize different stages & sexes
Practice picking worms;
Start Series 1: Set up autosomal and X-linked crosses
|3 days after lab: pick (2) wild type worms from each cross to new plates (3 plates total);
Examine the phenotypes of the progeny – hermaphrodites and males - record all information in your notebook
|Homework: Complete Entry Survey; Familiarize yourself with the information in the BISC219/F11:Resources section;|
Read all of the background information on C. elegans found in BISC219/F11:_Worm_Info and read the Lab 1 and Lab 2 information about our first Series 1: Autosomal vs. Sex-Linked Inheritance at BISC219/F11:_Gene_Linkage & BISC219/F11:_Lab_2; Complete Graded Assignment 1 explained at BISC219/F11: Assignment_1_Lab1
||Sept. 8 to
|Complete Series 1: Count and examine phenotypes of autosomal vs. X-linked crosses;
Start Series 2: Classical (Forward) Genetics- Part 1- Examine mutant worms and compare to wild type:
Pick (3) putative Dpy mutants to separate plates
|3 days after lab:Examine mutants: check phenotype
if Dpy then cross L4 mutant hermaphrodites by L4 N2 males
(2 plates – duplicate)
|Homework: Read ALL of Series 2:background information BISC219/F11:_Gene_Mapping_Info and about all work to be performed in Labs 2-6 on our Classical (Forward) Genetics project. Write a summary of our overall topic and experimental question(s) & goals. Outline the experimental process. Use this outline to write a 1-2 page summary of how you will determine the exact location and extent of the gene and protein change and include the significance of the mutation in worms and, if possible, broader significance in other species. Assignment explained and rubric found at BISC219/F11: Assignment Series2_Outline_Summary|
Start Data Analysis (Results) of your autosomal vs. X-linked testing DUE at the beginning of LAB 4.
Grading rubric & Assignment info at:
BISC219/F11: Assignment Help- Data Analysis 1
||Sept. 15 to
|Series 2- Part 2: Linkage Analysis: cross heterozygous males (+/d) from last cross to the (5) test strains (5 plates total);
|3 days after lab: Linkage: transfer (2) L4 hermaphrodites from each cross
to new plates (5 plates total)
|Homework: Draw crosses and diagram strategy for Series 2 Linkage Analysis due at the beginning of Lab 4. Information and grading rubric found at BISC219/F11: Assignment_Series2_Linkage Testing Crosses.
Con't Data Analysis (Results)of your autosomal vs. X-linked testing DUE at the beginning of LAB 4.
Grading rubric & Assignment info at:
BISC219/F11: Assignment Help- Data Analysis 1
||Sept. 22 to
|Complete Linkage Analysis: examine phenotypes and count to determine linkage;
Part 3: Start Mapping: pick (6) Uncs of the linked unc strain to separate plates (6 plates total);
Part 4: Start Complementation Analysis: Cross Dpy mutant worms to N2 males (2 plates total)
|3 days after lab: Mapping:
Pick (3) double mutants to separate plates (3 plates total);
Series2: Complementation: pick males from complementation cross #1 and mate with known Dpy strains (3 plates total);
|Homework: Explain complementation analysis in general and specifically how it was used to id your dpy gene and to determine if the mutation of interest has been previously characterized. Diagram complementation crosses using template provided. Due at the beginning of Lab 5. Assignment described atBISC219/F11: Assignment_Series2_Complementation
||Sept. 29 to
|Series2: Mapping:Self double mutants to keep viable true-breeding progeny;
Series2: Complementation: examine cross plates for Dpy males - WHY?; Series3: Reverse Genetics: Pick gene of interest; set up PCR reaction to clone the gene
|Series 3:Reverse Genetics: Examine the results of agarose gel electrophoresis
||Homework: Draw crosses and diagram strategy for Mapping the Location of your dpy mutation. (Complete template downloaded for Linkage analysis). Due at the beginning of Lab 6. Assignment described at BISC219/F11: Assignment_Series2_Mapping Crosses
||Oct. 12 to
|Series 2: Mapping: cross N2 males (++/++) with L4 double mutant hermaphrodites (2 plates total);Self double mutants to new plate
Series2: Characterizing the dpy mutation Gene Sequence Analysis
Series3: Reverse Genetics:Restriction enzyme digest PCR product/clean-up/ligation into pPD129.36 vector/transformation into BL21 E.coli
|3 days after lab:Series2: Mapping: pick (4) male heterozygotes (++/d u) and Cross them with L4 hermaphrodite double mutants (d u/d u).
Series 3:Check control & transformation plates (save) –
notify instructor if no colonies
|Homework: DNA sequencing Analysis due at the beginning of Lab 7. Assignment description at BISC219/F11: Assignment_Series2_DNA Sequencing|
||Oct. 19 to
|Series2:Finish Mapping: SCORE! Calculate Recombination frequency;
Series3:Reverse Genetics Colony PCR to look for bacterial transformants
|Determine map distance between your mutant gene
and the known reference mutation
Day before lab: Series 3:grow overnight culture of positive colony
|Homework: Scientific research report on Series 2 Classical (Forward) Genetics Project due Fri. Nov. 4 for ALL students. 5%/day late penalty! See assignment directions at: BISC219/F11: Assignment_ Series2_Classical Genetics Paper
||Nov. 2 to
|Series3: Reverse Genetics: Plasmid isolation/quantification of DNA/ transformation of plasmid into E. coli strain HT115(DE3)
||Day before lab: grow overnight culture of single colony from transformation
||Homework: Write all Series 3 Protocols as Materials & Methods. Assignment described at BISC219/F11: Assignment_Series3_ Materials and Methods. Due at the beginning of Lab 9.
||Nov. 9 to
|Series3:RNAi : Induction of bacteria and seed plates for RNAi feeding;
Journal article discussion
|4 days later: Pick 2 L4 hermaphrodite worms of N2 and rrf-3 genotype to 2 RNAi plates for each genotype and make 1 control mock plate for each genotype (6 plates);
||Homework:Write a draft introduction section (including properly formatted Literature Cited page) of your next paper on our Reverse Genetics Project. Due at the beginning of Lab 10. Refer to BISC219/F11:Resources Guide to Scientific Writing.
||Nov. 16 to
|Series3:RNAi: SCORING (collection)of phenotype of fed worms compared to control N2 worms & worms with gene mutation
||Homework: Construct figures/tables with properly formatted legends to illustrate the main findings of the RNAi part of your Series3:Reverse Genetics project. These figures are to be used in a Science Writing workshop in Lab 11. Full paper on Series 3 Due last day of classes for all students. Read the original 1998 paper by Fire and Mello published in Nature 391: 806-811 (Feb.19 1998); Do your own library search to find and read a recent review article about RNAi in C. elegans. Also read the 2002 Simmer et al. paper in Current Biology 12:1317-1319 explaining the rrf-3 strain that we will use for our RNAi work. These articles provide background information on how the mechanism of RNAi was worked out experimentally and will be helpful to you when you write the introduction section of your Series3 paper. Also read a published journal article by Green et al. (2009) Impact of Cigarette Smoke Exposure on Innate Immunity: A Caenorhabditis elegans Model PLoS ONE 4(8): e6860. This study, like yours, uses reverse genetics to investigate gene function.
||Nov. 23 to
||Nov. 28 to
|Series 3:Reverse Genetics Science Writing Workshop & Effective Figure Design. Be sure to have read the original 1998 paper by Fire and Mello published in Nature 391: 806-811 (Feb.19 1998); Do your own library search to find and read a recent review article about RNAi in C. elegans. Also read the 2002 Simmer et al. paper in Current Biology 12:1317-1319 explaining the rrf-3 strain that we will use for our RNAi work. These articles provide background information on how the mechanism of RNAi was worked out experimentally and will be helpful to you when you write the introduction section of your Series3 paper. Also read a published journal article by Green et al. (2009) Impact of Cigarette Smoke Exposure on Innate Immunity: A Caenorhabditis elegans Model PLoS ONE 4(8): e6860. This study, like yours, uses reverse genetics to investigate gene function.
||Homework: Full Scientific Research Report on Reverse Genetics Project – Title, Abstract, Intro, Materials and Methods, Results, Discussion, References See BISC219/F11:Resources section Guide to Scientific Wriing and BISC219/F11: Assignment_Series3_Reverse Genetics Paper using RNAi.
DUE Dec. 7 by 5 pm for ALL students. Complete exit genetic assessment. See Sakai site for link and for how to obtain your incentive points.