BISC219/F12: Lab 3 - Revision history

Melissa Beers: /* Lab 3: Forward Genetics Project: Linkage Analysis */

2012-09-18T18:09:28Z

Lab 3: Forward Genetics Project: Linkage Analysis

←Older revision		Revision as of 18:09, 18 September 2012
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	\| '''Chromosome 5''' \|\| ''unc-76'' \|\| u-shaped coiler		\| '''Chromosome 5''' \|\| ''unc-76'' \|\| u-shaped coiler
	\|-		\|-
-	~~\| '''X-Chromosome''' \|\| "unc-3" \|\| u-shaped coiler~~
	\|}		\|}

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	'''3-4 days after lab:'''		'''3-4 days after lab:'''

-	#For linkage testing, transfer 2 wild type cross progeny (potentially heterozygous for both traits) that are L4 stage hermaphrodites from each of your 6 crosses to each of 2 new plates per cross for a total of 12 plates.	+	#For linkage testing, transfer 2 wild type cross progeny (potentially heterozygous for both traits) that are L4 stage hermaphrodites from each of your 6 crosses to each of 2 new plates per cross for a total of 10 plates.
-	#Label your 12 (6 sets of duplicates) plates with your <font color=purple>PURPLE </font color=purple> Sharpie. Label each plate with your initials, the genotype of your worms and the date. In each case, why is it important that you transfer L4’s and not adults? What is the genotype and phenotype of your expected F2 progeny?	+	#Label your 10 (5 sets of duplicates) plates with your <font color=purple>PURPLE </font color=purple> Sharpie. Label each plate with your initials, the genotype of your worms and the date. In each case, why is it important that you transfer L4’s and not adults? What is the genotype and phenotype of your expected F2 progeny?
	#Incubate all worms at 23°C until the next lab period.		#Incubate all worms at 23°C until the next lab period.

Melissa Beers: /* Lab 3: Forward Genetics Project: Linkage Analysis */

2012-09-11T17:21:55Z

Lab 3: Forward Genetics Project: Linkage Analysis

←Older revision		Revision as of 17:21, 11 September 2012
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	'''To Do Today:'''		'''To Do Today:'''

-	1. For linkage testing set up ~~four~~ different crosses. Each cross will contain 3 heterozygous males (d/+; +/+) from the cross you initiated using your mutant Dpy worms. Make sure that these are the only animals that you transfer from that plate by transferring the males to a transfer plate and letting them crawl around for a minute - away from any contaminating worms - then pick a second time to the mating plate. <bR>	+	1. For linkage testing set up six different crosses. Each cross will contain 3 heterozygous males (d/+; +/+) from the cross you initiated using your mutant Dpy worms. Make sure that these are the only animals that you transfer from that plate by transferring the males to a transfer plate and letting them crawl around for a minute - away from any contaminating worms - then pick a second time to the mating plate. <bR>
-	2. Each heterozygous (d/+; +/+) male will be mated to three L4 hermaphrodites that are homozygous for one of 4 known ''unc''(+/+; u/u) mutations on a mating plate. The strains and their reference mutations are: <br>	+	2. Each heterozygous (d/+; +/+) male will be mated to three L4 hermaphrodites that are homozygous for one of 6 known ''unc''(+/+; u/u) mutations on a mating plate. The strains and their reference mutations are: <br>
	<center><br>		<center><br>

Line 23:		Line 23:
	\| '''Chromosome 1''' \|\| ''unc-13'' \|\| coiler		\| '''Chromosome 1''' \|\| ''unc-13'' \|\| coiler
	\|-		\|-
-	\| '''Chromosome 2''' \|\| ''unc-52'' \|\| ~~immobile~~	+	\| '''Chromosome 2''' \|\| ''unc-104'' \|\| coiler
	\|-		\|-
	\| '''Chromosome 3''' \|\| ''unc-32'' \|\| coiler		\| '''Chromosome 3''' \|\| ''unc-32'' \|\| coiler
Line 29:		Line 29:
	\| '''Chromosome 4''' \|\| ''unc-17'' \|\| coiler		\| '''Chromosome 4''' \|\| ''unc-17'' \|\| coiler
	\|-		\|-
		+	\| '''Chromosome 5''' \|\| ''unc-76'' \|\| u-shaped coiler
		+	\|-
		+	\| '''X-Chromosome''' \|\| "unc-3" \|\| u-shaped coiler
	\|}		\|}

	</center>		</center>

-	3. Label your ~~four~~ plates with your <font color=purple>PURPLE </font color=purple> Sharpie. With the genotype of the strain - for example: +/+; ''unc-13/unc-13'' (H) X d/+; +/+ (M) with your initials and date. <br>	+	3. Label your six plates with your <font color=purple>PURPLE </font color=purple> Sharpie. With the genotype of the strain - for example: +/+; ''unc-13/unc-13'' (H) X d/+; +/+ (M) with your initials and date. <br>
	4. Incubate all of the worms at 23°C for 3 days in your team's worm box.<br>		4. Incubate all of the worms at 23°C for 3 days in your team's worm box.<br>
	<br>		<br>
Line 39:		Line 42:
	'''3-4 days after lab:'''		'''3-4 days after lab:'''

-	#For linkage testing, transfer 2 wild type cross progeny (potentially heterozygous for both traits) that are L4 stage hermaphrodites from each of your 4 crosses to each of 2 new plates per cross for a total of 8 plates.	+	#For linkage testing, transfer 2 wild type cross progeny (potentially heterozygous for both traits) that are L4 stage hermaphrodites from each of your 6 crosses to each of 2 new plates per cross for a total of 12 plates.
-	#Label your 8 (4 sets of duplicates) plates with your <font color=purple>PURPLE </font color=purple> Sharpie. Label each plate with your initials, the genotype of your worms and the date. In each case, why is it important that you transfer L4’s and not adults? What is the genotype and phenotype of your expected F2 progeny?	+	#Label your 12 (6 sets of duplicates) plates with your <font color=purple>PURPLE </font color=purple> Sharpie. Label each plate with your initials, the genotype of your worms and the date. In each case, why is it important that you transfer L4’s and not adults? What is the genotype and phenotype of your expected F2 progeny?
	#Incubate all worms at 23°C until the next lab period.		#Incubate all worms at 23°C until the next lab period.

Tucker Crum: /* Assignment */

2012-08-28T17:15:43Z

Assignment

←Older revision		Revision as of 17:15, 28 August 2012
Line 44:		Line 44:

	== Assignment ==		== Assignment ==
-	Remember to check the [[BISC219/F12:Assignments \| Assignment section]] of the wiki for instructions about the graded assignment due in the next lab and check the Weekly Planner section of the [[BISC219/F12:Calendars/Planner\| ~~Weekly~~ Calendar]] for other work to accomplish before the next lab. [http://www.openwetware.org/index.php?title=BISC219/F12:_Assignment_Series2_Linkage_Testing_Crosses&action=edit Lab 3 homework link]	+	Remember to check the [[BISC219/F12:Assignments \| Assignment section]] of the wiki for instructions about the graded assignment due in the next lab and check the '''Weekly Planner''' section of the [[BISC219/F12:Calendars/Planner\| Calendar/ Weekly Lab Planner]] for other work to accomplish before the next lab. [http://www.openwetware.org/index.php?title=BISC219/F12:_Assignment_Series2_Linkage_Testing_Crosses&action=edit Lab 3 homework link]
	<div class=noprint>		<div class=noprint>

Tucker Crum: /* Assignment */

2012-08-28T16:05:48Z

Assignment

←Older revision		Revision as of 16:05, 28 August 2012
Line 44:		Line 44:

	== Assignment ==		== Assignment ==
-	Remember to check the [~~http://www.openwetware.org/wiki/~~BISC219/F12:Assignments 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. [http://www.openwetware.org/index.php?title=BISC219/F12:_Assignment_Series2_Linkage_Testing_Crosses&action=edit Lab 3 homework link]	+	Remember to check the [[BISC219/F12:Assignments \| Assignment section]] of the wiki for instructions about the graded assignment due in the next lab and check the Weekly Planner section of the [[BISC219/F12:Calendars/Planner\| Weekly Calendar]] for other work to accomplish before the next lab. [http://www.openwetware.org/index.php?title=BISC219/F12:_Assignment_Series2_Linkage_Testing_Crosses&action=edit Lab 3 homework link]
	<div class=noprint>		<div class=noprint>

Tucker Crum: /* Links to Labs& Project Info */

2012-08-22T14:58:00Z

Links to Labs& Project Info

←Older revision		Revision as of 14:58, 22 August 2012
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	[[BISC219/F12: Lab 7 \| Lab 7: Complete Mapping: Score]]<br>		[[BISC219/F12: Lab 7 \| Lab 7: Complete Mapping: Score]]<br>
	'''Series3:'''<BR>		'''Series3:'''<BR>
-		+	[[BISC219/F12: RNAi General Information\| Background Information on Project 3: Investigating Gene Regulation Using RNAi]] <br>
		+	[[BISC219/F12: Media Recipes \| Media Recipes]]<br>
		+	[[BISC219/F12: RNAi Lab 7 \| Lab 7: Identifying a bacterial colony containing our plasmid of interest ]]<br>
		+	[[BISC219/F12: RNAi Lab 8 \| Lab 8: Creating the feeding strain of bacteria for RNAi]]<br>
		+	[[BISC219/F12: RNAi Lab 9 \| Lab 9: Induction of feeding strain to produce dsRNA and feeding worms]]<br>
		+	[[BISC219/F12: RNAi Lab 10 \| Lab 10: Phenotypic analysis of treated vs untreated worms]]<br>
		+	[[BISC219/F12: RNAi Lab 11 \| Lab 11: Writing Workshop]]<br>
		+	[[BISC219/F12: RNAi Lab 12 \| Lab 12: Writing Conferences]]<br>
	</div>		</div>

Tucker Crum: /* Links to Labs& Project Info */

2012-08-20T19:35:56Z

Links to Labs& Project Info

←Older revision		Revision as of 19:35, 20 August 2012
Line 61:		Line 61:
	[[BISC219/F12: Lab 7 \| Lab 7: Complete Mapping: Score]]<br>		[[BISC219/F12: Lab 7 \| Lab 7: Complete Mapping: Score]]<br>
	'''Series3:'''<BR>		'''Series3:'''<BR>
-	~~[[BISC219/F12:RNA interference \| RNA interference]]<BR>~~	+
-	~~[[BISC219/F12: RNAi General Information \| RNAi General Information]] <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 6 \| Lab 6: Cloning your gene of interest]]<br>~~	+
-	~~[[BISC219/F12: RNAi Lab 7 \| Lab 7: Picking your transformant]]<br>~~	+
-	~~[[BISC219/F12: RNAi Lab 8 \| Lab 8: Plasmid purification and transformation]]<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>~~	+
	</div>		</div>

Melissa Beers: /* Assignment */

2012-08-16T14:43:37Z

Assignment

←Older revision		Revision as of 14:43, 16 August 2012
Line 44:		Line 44:

	== Assignment ==		== Assignment ==
-	Remember to check the [http://www.openwetware.org/wiki/BISC219/F12:Assignments 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. [http://www.openwetware.org/index.php?title=BISC219/~~F11~~:_Assignment_Series2_Linkage_Testing_Crosses&action=edit Lab 3 homework link]	+	Remember to check the [http://www.openwetware.org/wiki/BISC219/F12:Assignments 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. [http://www.openwetware.org/index.php?title=BISC219/F12:_Assignment_Series2_Linkage_Testing_Crosses&action=edit Lab 3 homework link]
	<div class=noprint>		<div class=noprint>

Melissa Beers: /* Assignment */

2012-08-16T14:43:17Z

Assignment

←Older revision		Revision as of 14:43, 16 August 2012
Line 44:		Line 44:

	== 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 [http://www.openwetware.org/wiki/BISC219/F12:Assignments 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. [http://www.openwetware.org/index.php?title=BISC219/F11:_Assignment_Series2_Linkage_Testing_Crosses&action=edit Lab 3 homework link]
	<div class=noprint>		<div class=noprint>
		+
	==Links to Labs& Project Info==		==Links to Labs& Project Info==
	'''Series1:'''<BR>		'''Series1:'''<BR>

Tucker Crum at 13:32, 9 May 2012

2012-05-09T13:32:28Z

←Older revision		Revision as of 13:32, 9 May 2012
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	{{Template:BISC219/F12}}		{{Template:BISC219/F12}}
-	<div style="padding: 10px; width: 720px; border: 5px solid #~~6600CC~~;">	+	<div style="padding: 10px; width: 720px; border: 5px solid #7E354D;;">

	== Lab 3: Forward Genetics Project: Linkage Analysis ==		== Lab 3: Forward Genetics Project: Linkage Analysis ==

Tucker Crum: New page: {{Template:BISC219/F12}}
== Lab 3: Forward Genetics Project: Linkage Analysis == Now that you have found evidence of ...

2012-05-09T13:31:35Z

New page: {{Template:BISC219/F12}} <div style="padding: 10px; width: 720px; border: 5px solid #6600CC;"> == Lab 3: Forward Genetics Project: Linkage Analysis == Now that you have found evidence of ...

New page

{{Template:BISC219/F12}}
<div style="padding: 10px; width: 720px; border: 5px solid #6600CC;">

== Lab 3: Forward Genetics Project: Linkage Analysis ==
Now that you have found evidence of a homozygous recessive gene defect that is associated with a phenotype alteration from wild type (true breeding mutant), you will determine on which chromosome this defective gene is located. First, you need to determine whether or not the mutation is x-linked and, if not, on which of the five autosomes (possible linkage groups) it is found. This task is a prerequisite to mapping the mutation (locating where on a particular chromosome the mutation is likely to be found). Linkage testing is accomplished by determining the segregation behavior of your unmapped mutation relative to standard reference markers (e.g., mutations whose locations are already known). Recall that unlinked genes will segregate independently (your basic dihybrid inheritance as first observed by Gregor Mendel) whereas mutations associated with linked genes will not.

In practice, linkage tests are performed using the following steps (where "d" (''dpy'') represents your recessive mutant tested with reference marker "u" (''unc'')). The markers d and u must be visually distinguishable. Since homozygous mutant males usually will not mate, the desired double heterozygote is constructed by mating males that are heterozygous for your ''dpy'' mutation [wild type for all other genes including the reference mutation (d/+;+/+)] with hermaphrodites that are homozygous for the reference mutation ''unc'' (+/+; u/u) and have no ''dpy'' mutation. The genotypes of the F1 hybrids will be (+/d;u/+) and (+/+;u/+). We are only interested in the double heterozygote (+/d;u/+). The F1 hybrids containing only u are not useful. To select the (+/d;u/+) heterozygotes, we let 2 F1's self fertilize on two separate plates (two animal on each plate for a total of 4 worms). We score the progeny of the F1 individuals (the F2) for linkage. Only F1 worms which produce d/d homozygotes in the F2 generation are scored, since those are the F1 (+/d;u/+). You should find d/d homozygotes on 50% of the plates. Why?

F2 progeny of each class are counted in the (+/d;u/+) plates: wild-type (+/+;+/+); d (d/d;+/+); u (+/+;u/u) and du double (d/d;u/u). If assortment is independent, progeny will be: 9/16 wild type; 3/16 d, 3/16 u; 1/16 du (that is the 9:3:3:1 ratio)!

On the other hand, if the markers are closely linked double homozygotes (d u/d u) will occur only through a very rare recombination event; therefore, you are not likely to observe the double mutant class.

'''To Do Today:'''

1. For linkage testing set up four different crosses. Each cross will contain 3 heterozygous males (d/+; +/+) from the cross you initiated using your mutant Dpy worms. Make sure that these are the only animals that you transfer from that plate by transferring the males to a transfer plate and letting them crawl around for a minute - away from any contaminating worms - then pick a second time to the mating plate. 
2. Each heterozygous (d/+; +/+) male will be mated to three L4 hermaphrodites that are homozygous for one of 4 known ''unc''(+/+; u/u) mutations on a mating plate. The strains and their reference mutations are: 
<center> 

{| border="1"
|+
! Chromosome !! Strain !! Phenotype
|-
| '''Chromosome 1''' || ''unc-13'' || coiler
|-
| '''Chromosome 2''' || ''unc-52'' || immobile
|-
| '''Chromosome 3''' || ''unc-32'' || coiler
|-
| '''Chromosome 4''' || ''unc-17'' || coiler
|-
|}

</center>

3. Label your four plates with your PURPLE Sharpie. With the genotype of the strain - for example: +/+; ''unc-13/unc-13'' (H) X d/+; +/+ (M) with your initials and date. 
4. Incubate all of the worms at 23°C for 3 days in your team's worm box. 
 

'''3-4 days after lab:'''

#For linkage testing, transfer 2 wild type cross progeny (potentially heterozygous for both traits) that are L4 stage hermaphrodites from each of your 4 crosses to each of 2 new plates per cross for a total of 8 plates.
#Label your 8 (4 sets of duplicates) plates with your PURPLE Sharpie. Label each plate with your initials, the genotype of your worms and the date. In each case, why is it important that you transfer L4’s and not adults? What is the genotype and phenotype of your expected F2 progeny?
#Incubate all worms at 23°C until the next lab period.

== 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>
==Links to Labs& Project Info==
'''Series1:''' 
[[BISC219/F12: Worm Info | Worm Info]] 
[[BISC219/F12: Gene Linkage | Lab 1: Worm Boot Camp & Sex-Linked or Autosomal Start]] 
[[BISC219/F12: Lab 2 | Lab 2: Sex-Linked or Autosomal Finale]] 
'''Series2:''' 
[[BISC219/F12: Gene Mapping Info | Background: Classical Forward Genetics and Gene Mapping]] 
[[BISC219/F12: Lab 2 Mutant Hunt | Lab 2: Mutant Hunt]] 
[[BISC219/F12: Lab 3 | Lab 3: Linkage Test Part 1]] 
[[BISC219/F12: Lab 4 | Lab 4: Linkage Test Part 2, Mapping and Complementation]] 
[[BISC219/F12: Lab 5 | Lab 5: Finish Complementation; Mapping Continued]] 
[[BISC219/F12: Lab 6 | Lab 6: DNA sequence analysis; Mapping Continued]] 
[[BISC219/F12: Lab 7 | Lab 7: Complete Mapping: Score]] 
'''Series3:''' 
[[BISC219/F12:RNA interference | RNA interference]] 
[[BISC219/F12: RNAi General Information | RNAi General Information]] 
[[BISC219/F12: Media Recipes | Media Recipes]] 
[[BISC219/F12: RNAi Lab 5 | Lab 5: Picking your gene to RNAi]] 
[[BISC219/F12: RNAi Lab 6 | Lab 6: Cloning your gene of interest]] 
[[BISC219/F12: RNAi Lab 7 | Lab 7: Picking your transformant]] 
[[BISC219/F12: RNAi Lab 8 | Lab 8: Plasmid purification and transformation]] 
[[BISC219/F12: RNAi Lab 9 | Lab 9: Induction of bacteria for RNAi]] 
[[BISC219/F12: RNAi Lab 10 | Lab 10: Scoring your worms]] 
[[BISC219/F12: RNAi Lab 11 | Lab 11: ]] 
</div>

←Older revision		Revision as of 14:58, 22 August 2012
Line 61:		Line 61:
	[[BISC219/F12: Lab 7 \| Lab 7: Complete Mapping: Score]]<br>		[[BISC219/F12: Lab 7 \| Lab 7: Complete Mapping: Score]]<br>
	'''Series3:'''<BR>		'''Series3:'''<BR>
-		+	[[BISC219/F12: RNAi General Information\| Background Information on Project 3: Investigating Gene Regulation Using RNAi]] <br>
		+	[[BISC219/F12: Media Recipes \| Media Recipes]]<br>
		+	[[BISC219/F12: RNAi Lab 7 \| Lab 7: Identifying a bacterial colony containing our plasmid of interest ]]<br>
		+	[[BISC219/F12: RNAi Lab 8 \| Lab 8: Creating the feeding strain of bacteria for RNAi]]<br>
		+	[[BISC219/F12: RNAi Lab 9 \| Lab 9: Induction of feeding strain to produce dsRNA and feeding worms]]<br>
		+	[[BISC219/F12: RNAi Lab 10 \| Lab 10: Phenotypic analysis of treated vs untreated worms]]<br>
		+	[[BISC219/F12: RNAi Lab 11 \| Lab 11: Writing Workshop]]<br>
		+	[[BISC219/F12: RNAi Lab 12 \| Lab 12: Writing Conferences]]<br>
	</div>		</div>

←Older revision		Revision as of 19:35, 20 August 2012
Line 61:		Line 61:
	[[BISC219/F12: Lab 7 \| Lab 7: Complete Mapping: Score]]<br>		[[BISC219/F12: Lab 7 \| Lab 7: Complete Mapping: Score]]<br>
	'''Series3:'''<BR>		'''Series3:'''<BR>
-	~~[[BISC219/F12:RNA interference \| RNA interference]]<BR>~~	+
-	~~[[BISC219/F12: RNAi General Information \| RNAi General Information]] <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 6 \| Lab 6: Cloning your gene of interest]]<br>~~	+
-	~~[[BISC219/F12: RNAi Lab 7 \| Lab 7: Picking your transformant]]<br>~~	+
-	~~[[BISC219/F12: RNAi Lab 8 \| Lab 8: Plasmid purification and transformation]]<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>~~	+
	</div>		</div>

BISC219/F12: Lab 3 - Revision history

Melissa Beers: /* Lab 3: Forward Genetics Project: Linkage Analysis */

Melissa Beers: /* Lab 3: Forward Genetics Project: Linkage Analysis */

Tucker Crum: /* Assignment */

Tucker Crum: /* Assignment */

Tucker Crum: /* Links to Labs& Project Info */

Tucker Crum: /* Links to Labs& Project Info */

Melissa Beers: /* Assignment */

Melissa Beers: /* Assignment */

Tucker Crum at 13:32, 9 May 2012

Tucker Crum: New page: {{Template:BISC219/F12}} == Lab 3: Forward Genetics Project: Linkage Analysis == Now that you have found evidence of ...

Tucker Crum: New page: {{Template:BISC219/F12}}
== Lab 3: Forward Genetics Project: Linkage Analysis == Now that you have found evidence of ...