BISC219/F13: Lab 2

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#Examine each plate of F2 progeny.  If you chose only L4 hermaphrodites, as instructed,you should only see hermaphrodite progeny.  If you have a lot of males on your plates, you probably chose young adult worms rather than L4 hermaphrodite's. That's a problem - see your instructor.<br>
#Examine each plate of F2 progeny.  If you chose only L4 hermaphrodites, as instructed,you should only see hermaphrodite progeny.  If you have a lot of males on your plates, you probably chose young adult worms rather than L4 hermaphrodite's. That's a problem - see your instructor.<br>
#For each cross, you should count and examine a random sample of 100 worms. The mutant worms may be smaller and not move as well as the wild type worms.  Look around your plate to get a quick assessment of the population.<br>
#For each cross, you should count and examine a random sample of 100 worms. The mutant worms may be smaller and not move as well as the wild type worms.  Look around your plate to get a quick assessment of the population.<br>
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#Record in your lab notebook the number of WT, Dpy, Unc, or Dpy Unc mutants by examining the phenotype as you remove each animal from the plate. (Be sure to flame the pick after removing each worm!!!) If the genes responsible for the mutations are unlinked, you should see WT's (+/+;+/+), Dpy’s (''dpy/dpy'';+/+), Unc’s(+/+;''unc/unc'') and Dpy Unc’s (''d/d;u/u'') in a ratio of 9:3:3:1.  If linked, you should see a greater proportion than expected of  Dpy Unc’s (''d u/d u'') double mutants vs Dpy or Unc single mutants among the mutant hermaphrodite progeny.<br>
+
#Record in your lab notebook the number of WT, Dpy, Unc, or Dpy Unc mutants by examining the phenotype as you remove each animal from the plate. (Be sure to flame the pick after removing each worm!!!)  
-
The main challenge is to correctly differentiate single mutants of each phenotype (Dpy or Unc) from double mutants(Dpy AND Unc). Students tend to undercount single mutants and overcount doubles when they are inexperienced. Check with your instructor if you are unsure about how to score these groups. WT worms may be undercounted because they are harder to "catch". Be careful not to skew your data in this way. Pick what you consider single mutants to a plate and what you consider double mutants to another plate.  Ask your instructor to check them before you go to far in the scoring process.
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<br>
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If the genes responsible for the mutations are unlinked, you should see WT's (+/+;+/+), Dpy’s (''dpy/dpy'';+/+), Unc’s(+/+;''unc/unc'') and Dpy Unc’s (''d/d;u/u'') in a ratio of 9 WT:3 Dpy:3 Unc:1 Dpy Unc.  If linked, you should see a greater proportion than expected of  Dpy Unc’s (''d u/d u'') double mutants and fewer Dpy or Unc single mutants among the mutant hermaphrodite progeny.<br>
 +
<br>
 +
The main challenge is to correctly differentiate single mutants of each phenotype (Dpy or Unc) from double mutants (Dpy AND Unc). Students tend to undercount single mutants and overcount doubles when they are inexperienced. Check with your instructor if you are unsure about how to score these groups. WT worms may be undercounted because they are harder to "catch". Be careful not to skew your data in this way. Pick what you consider single mutants to a plate and what you consider double mutants to another plate.  Ask your instructor to check them before you go to far in the scoring process.
'''Make sure you copy your data into the appropriate place on the course spreadsheet!'''
'''Make sure you copy your data into the appropriate place on the course spreadsheet!'''
<br><br>
<br><br>
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You should now be able to conclude which strain has mutations that are autosomal and linked, which strain has mutations that are both autosomal and unlinked, and which strain has an autosomal mutation and an x-linked mutation responsible for either the Dpy or Unc phenotype (which one?).<br>
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You should now be able make educated conclusions about the inheritance of ''dpy'' and ''unc'' genes in these strains. 
 +
<br>
 +
<br>
 +
Among the possibilities are the following:
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#Both mutations on the same autosome (linked).
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#The two mutations are on different autosomes (unlinked).
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#One mutation is on an autosome and the other is on the sex chromosome (unlinked).
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#Both mutations are on the sex chromosome (linked)<br>
<br>
<br>
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== Lab 2: Start Forward Genetics Project 2: Testing a Mutant for True Breeding ==
 +
Make sure you have read the background information about Project 2 found at [http://openwetware.org/wiki/BISC219/F13:Gene_Mapping | Background Project 2 Forward Genetics Investigation]<bR>
 +
'''To Do Today (per group)'''<br>
 +
#Choose one of the six plates of "mutagenized" worms at the front of the room <br>
 +
#Scan the “mutagenized” worms on this plate and determine their phenotype.  Make sure you write this down.
 +
#Transfer 1 putative mutant hermaphrodite to each of two new plates.  Take care not to transfer any other animals or eggs other than your putative mutant.<br>
 +
#Label these 2 identical plates with the hermaphrodite mutant's strain information and your group's information (team color, initials, lab day, date). Use your <font color= blue><b>BLUE </b></font color> Sharpie for labeling these linkage analysis plates.  Labels belong on the side of the plate with the media in it. Tape makes it hard to see the worms on later days.  '''DO NOT write on the lids''' - they fall off and you will not know what cover goes to what bottom!
 +
#Put an elastic around the two phenotype determination plates. Place them in your group's plastic box (make sure your box is labeled with your names and lab section on a piece of your team color tape).
 +
#Place your box on the shelf in the incubator for your lab section.<br>
 +
#Incubate your worms at 23°C for 3 days.<br>
 +
<br>
 +
'''3 Days After Lab'''<br>
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#Examine your two plates containing your mutant worms and their F1 progeny.  Are all the worms on at least one plate of the Dpy phenotype?  If '''YES''', then you are good to go on with the next cross. If you don't have all dumpy mutants in the F1 generation on a plate, you should '''NOT''' use that plate to select worms for the next cross. If neither of your plates contains all dumpy mutants, email your instructor ASAP!<br>
 +
# If you have an appropriate plate for continuing: Set up a pair of replicate crosses between 4  Dpy L4 hermaphrodites (should still have the vulval clearing about 1/2 way down the body of the worm) and 3-4 wild type male worms. You will have to search to find the wild type male worms among the hermaphrodites in the wild type worm plates provided in your lab day's box at the back of the room. When you are finished, you should have two identical mating plates with 3-4 L4 hermaphrodites and 3-4 wild type males.<br>
 +
#Label these identical plates with the hermaphrodite mutant's strain information and your group's information including the date. Use your <font color= blue><b>BLUE </b></font color> Sharpie for labeling these linkage analysis/mapping plates. <br>
 +
#Incubate your worms at 23°C until next lab period.
== Assignment ==
== Assignment ==
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See [http://openwetware.org/wiki/BISC219/F13:_Assignment_Help-_Data_Analysis_1 | Assignment_Help-_Data_Analysis_1] for instructions on Series 1 Results section write up for this project. Due at the beginning of Lab 3.'''   
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Find a rubric and more information about the assignment due at the beginning of Lab 3 at: [[BISC219/F13: Assignment_Series2_Outline_Summary | Assignment Series 2 Summary & Linkage Testing Description]]. Due at the beginning of Lab 3. <BR><BR>
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See [http://openwetware.org/wiki/BISC219/F13:_Assignment_Help-_Data_Analysis_1 | Assignment_Help-_Data_Analysis_1] for instructions on Series 1 Results section write up for this project. Due at the beginning of Lab 4.'''  <BR>
<div class=noprint>
<div class=noprint>
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==Links to Labs& Project Info==
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{{Template:BISC219/F13:Lab_Links}}
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'''Series1:'''<BR>
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[[BISC219/F13: Worm Info | Worm Info]] <br>
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[[BISC219/F13: Gene Linkage | Lab 1: Worm Boot Camp & Sex-Linked or Autosomal Start]]<BR>
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[[BISC219/F13: Lab 2 | Lab 2: Sex-Linked or Autosomal Finale]]<br>
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'''Series2:'''<BR>
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[[BISC219/F13: Gene Mapping Info | Background: Classical Forward Genetics and Gene Mapping]]<br>
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[[BISC219/F13: Lab 2 Mutant Hunt | Lab 2: Mutant Hunt]]<br>
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[[BISC219/F13: Lab 3  | Lab 3: Linkage Test Part 1]]<br>
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[[BISC219/F13: Lab 4  | Lab 4: Linkage Test Part 2, Mapping and Complementation]]<br>
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[[BISC219/F13: Lab 5  | Lab 5: Finish Complementation; Mapping Continued]]<br>
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[[BISC219/F13: Lab 6 | Lab 6: DNA sequence analysis; Mapping Continued]]<BR>
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[[BISC219/F13: Lab 7  | Lab 7: Complete Mapping: Score]]<br>
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'''Series3:'''<BR>
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[[BISC219/F13: RNAi General Information| Background Information on Project 3: Investigating Gene Regulation Using RNAi]] <br>
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[[BISC219/F13: Media Recipes | Media Recipes]]<br>
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[[BISC219/F13: RNAi Lab 7  | Lab 7: Identifying a bacterial colony containing our plasmid of interest  ]]<br>
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[[BISC219/F13: RNAi Lab 8  | Lab 8: Creating the feeding strain of bacteria for RNAi]]<br>
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[[BISC219/F13: RNAi Lab 9  | Lab 9: Induction of feeding strain to produce dsRNA and feeding worms]]<br>
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[[BISC219/F13: RNAi Lab 10 | Lab 10: Phenotypic analysis of treated vs untreated worms]]<br>
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[[BISC219/F13: RNAi Lab 11 | Lab 11: Writing Workshop]]<br>
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[[BISC219/F13: RNAi Lab 12 | Lab 12: Writing Conferences]]<br>
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</div>
</div>

Current revision

Contents

Lab 2: Finish Project 1- Autosomal or Sex-linked Inheritance?

  1. Examine each plate of F2 progeny. If you chose only L4 hermaphrodites, as instructed,you should only see hermaphrodite progeny. If you have a lot of males on your plates, you probably chose young adult worms rather than L4 hermaphrodite's. That's a problem - see your instructor.
  2. For each cross, you should count and examine a random sample of 100 worms. The mutant worms may be smaller and not move as well as the wild type worms. Look around your plate to get a quick assessment of the population.
  3. Record in your lab notebook the number of WT, Dpy, Unc, or Dpy Unc mutants by examining the phenotype as you remove each animal from the plate. (Be sure to flame the pick after removing each worm!!!)


If the genes responsible for the mutations are unlinked, you should see WT's (+/+;+/+), Dpy’s (dpy/dpy;+/+), Unc’s(+/+;unc/unc) and Dpy Unc’s (d/d;u/u) in a ratio of 9 WT:3 Dpy:3 Unc:1 Dpy Unc. If linked, you should see a greater proportion than expected of Dpy Unc’s (d u/d u) double mutants and fewer Dpy or Unc single mutants among the mutant hermaphrodite progeny.

The main challenge is to correctly differentiate single mutants of each phenotype (Dpy or Unc) from double mutants (Dpy AND Unc). Students tend to undercount single mutants and overcount doubles when they are inexperienced. Check with your instructor if you are unsure about how to score these groups. WT worms may be undercounted because they are harder to "catch". Be careful not to skew your data in this way. Pick what you consider single mutants to a plate and what you consider double mutants to another plate. Ask your instructor to check them before you go to far in the scoring process. Make sure you copy your data into the appropriate place on the course spreadsheet!

You should now be able make educated conclusions about the inheritance of dpy and unc genes in these strains.

Among the possibilities are the following:

  1. Both mutations on the same autosome (linked).
  2. The two mutations are on different autosomes (unlinked).
  3. One mutation is on an autosome and the other is on the sex chromosome (unlinked).
  4. Both mutations are on the sex chromosome (linked)


Lab 2: Start Forward Genetics Project 2: Testing a Mutant for True Breeding

Make sure you have read the background information about Project 2 found at | Background Project 2 Forward Genetics Investigation
To Do Today (per group)

  1. Choose one of the six plates of "mutagenized" worms at the front of the room
  2. Scan the “mutagenized” worms on this plate and determine their phenotype. Make sure you write this down.
  3. Transfer 1 putative mutant hermaphrodite to each of two new plates. Take care not to transfer any other animals or eggs other than your putative mutant.
  4. Label these 2 identical plates with the hermaphrodite mutant's strain information and your group's information (team color, initials, lab day, date). Use your BLUE Sharpie for labeling these linkage analysis plates. Labels belong on the side of the plate with the media in it. Tape makes it hard to see the worms on later days. DO NOT write on the lids - they fall off and you will not know what cover goes to what bottom!
  5. Put an elastic around the two phenotype determination plates. Place them in your group's plastic box (make sure your box is labeled with your names and lab section on a piece of your team color tape).
  6. Place your box on the shelf in the incubator for your lab section.
  7. Incubate your worms at 23°C for 3 days.


3 Days After Lab

  1. Examine your two plates containing your mutant worms and their F1 progeny. Are all the worms on at least one plate of the Dpy phenotype? If YES, then you are good to go on with the next cross. If you don't have all dumpy mutants in the F1 generation on a plate, you should NOT use that plate to select worms for the next cross. If neither of your plates contains all dumpy mutants, email your instructor ASAP!
  2. If you have an appropriate plate for continuing: Set up a pair of replicate crosses between 4 Dpy L4 hermaphrodites (should still have the vulval clearing about 1/2 way down the body of the worm) and 3-4 wild type male worms. You will have to search to find the wild type male worms among the hermaphrodites in the wild type worm plates provided in your lab day's box at the back of the room. When you are finished, you should have two identical mating plates with 3-4 L4 hermaphrodites and 3-4 wild type males.
  3. Label these identical plates with the hermaphrodite mutant's strain information and your group's information including the date. Use your BLUE Sharpie for labeling these linkage analysis/mapping plates.
  4. Incubate your worms at 23°C until next lab period.

Assignment

Find a rubric and more information about the assignment due at the beginning of Lab 3 at: Assignment Series 2 Summary & Linkage Testing Description. Due at the beginning of Lab 3.

See | Assignment_Help-_Data_Analysis_1 for instructions on Series 1 Results section write up for this project. Due at the beginning of Lab 4.

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