McClean: Tetrad Dissection - Revision history

Megan N McClean: /* Dissection */

Megan N McClean — Thu, 03 Nov 2011 13:56:21 GMT

Dissection

←Older revision		Revision as of 13:56, 3 November 2011
Line 58:		Line 58:
	## Move the microscope stage to the left or right 5 mm (one click) from the line of the four spores, and select another four-spore cluster. Separate the spores as before by 5-mm intervals. By this method, 10 tetrads can be dissected on each side of the YPD plate.		## Move the microscope stage to the left or right 5 mm (one click) from the line of the four spores, and select another four-spore cluster. Separate the spores as before by 5-mm intervals. By this method, 10 tetrads can be dissected on each side of the YPD plate.
	## You can use the following "record keeping tables" for keeping track of tetrads that have been put down correctly, to make notes about dropped tetrads, etc. The black line in the middle of each table represents you initial streak of digested cells in the center of the plate.		## You can use the following "record keeping tables" for keeping track of tetrads that have been put down correctly, to make notes about dropped tetrads, etc. The black line in the middle of each table represents you initial streak of digested cells in the center of the plate.
-	##* Record keeping table in .docx format: [[Tetrad_Dissection_Record_Keeping_Tables.docx \| Tetrad Record Keeping Table (docx)]]	+	##* Record keeping table in .docx format: [[Media:Tetrad_Dissection_Record_Keeping_Tables.docx \| Tetrad Record Keeping Table (docx)]]
-	##* Record keeping table in PDF format: [[Tetrad_Dissection_Record_Keeping_Tables.pdf \| Tetrad Record Keeping Table (PDF)]]	+	##* Record keeping table in PDF format: [[Media:Tetrad_Dissection_Record_Keeping_Tables.pdf \| Tetrad Record Keeping Table (PDF)]]
	## Remove the YPD plate from the stage, taking care not to break the microneedle.		## Remove the YPD plate from the stage, taking care not to break the microneedle.
	# Incubate the plate for 2-3 days at room temperature.		# Incubate the plate for 2-3 days at room temperature.

Megan N McClean: /* Dissection */

Megan N McClean — Thu, 03 Nov 2011 13:52:55 GMT

Dissection

←Older revision		Revision as of 13:52, 3 November 2011
Line 58:		Line 58:
	## Move the microscope stage to the left or right 5 mm (one click) from the line of the four spores, and select another four-spore cluster. Separate the spores as before by 5-mm intervals. By this method, 10 tetrads can be dissected on each side of the YPD plate.		## Move the microscope stage to the left or right 5 mm (one click) from the line of the four spores, and select another four-spore cluster. Separate the spores as before by 5-mm intervals. By this method, 10 tetrads can be dissected on each side of the YPD plate.
	## You can use the following "record keeping tables" for keeping track of tetrads that have been put down correctly, to make notes about dropped tetrads, etc. The black line in the middle of each table represents you initial streak of digested cells in the center of the plate.		## You can use the following "record keeping tables" for keeping track of tetrads that have been put down correctly, to make notes about dropped tetrads, etc. The black line in the middle of each table represents you initial streak of digested cells in the center of the plate.
-	##* Record keeping table in .docx format: [[Tetrad ~~Dissection~~ Record Keeping ~~Tables.~~docx]]	+	##* Record keeping table in .docx format: [[Tetrad_Dissection_Record_Keeping_Tables.docx \| Tetrad Record Keeping Table (docx)]]
		+	##* Record keeping table in PDF format: [[Tetrad_Dissection_Record_Keeping_Tables.pdf \| Tetrad Record Keeping Table (PDF)]]
	## Remove the YPD plate from the stage, taking care not to break the microneedle.		## Remove the YPD plate from the stage, taking care not to break the microneedle.
	# Incubate the plate for 2-3 days at room temperature.		# Incubate the plate for 2-3 days at room temperature.

Megan N McClean: /* Dissection */

Megan N McClean — Thu, 03 Nov 2011 13:50:21 GMT

Dissection

←Older revision		Revision as of 13:50, 3 November 2011
Line 57:		Line 57:
	## Pick up the remaining spore. Move the stage 5 mm (one click) and deposit the remaining spore.		## Pick up the remaining spore. Move the stage 5 mm (one click) and deposit the remaining spore.
	## Move the microscope stage to the left or right 5 mm (one click) from the line of the four spores, and select another four-spore cluster. Separate the spores as before by 5-mm intervals. By this method, 10 tetrads can be dissected on each side of the YPD plate.		## Move the microscope stage to the left or right 5 mm (one click) from the line of the four spores, and select another four-spore cluster. Separate the spores as before by 5-mm intervals. By this method, 10 tetrads can be dissected on each side of the YPD plate.
		+	## You can use the following "record keeping tables" for keeping track of tetrads that have been put down correctly, to make notes about dropped tetrads, etc. The black line in the middle of each table represents you initial streak of digested cells in the center of the plate.
		+	##* Record keeping table in .docx format: [[Tetrad Dissection Record Keeping Tables.docx]]
	## Remove the YPD plate from the stage, taking care not to break the microneedle.		## Remove the YPD plate from the stage, taking care not to break the microneedle.
	# Incubate the plate for 2-3 days at room temperature.		# Incubate the plate for 2-3 days at room temperature.

Megan N McClean: /* Notes */

Megan N McClean — Thu, 03 Nov 2011 13:44:49 GMT

Notes

←Older revision		Revision as of 13:44, 3 November 2011
Line 62:		Line 62:
	==Notes==		==Notes==
	<!-- Please paste this section "as is" into your protocol, and add notes to it if you have some!-->		<!-- Please paste this section "as is" into your protocol, and add notes to it if you have some!-->
-	Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!	+	Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
	#'''*[[User:Megan N McClean\|Megan N McClean]]''' When I can't age my dissection plates on my bench for a few days, I will stick them in the 30°C or 37°C warm room the morning of the day I dissect to dry them out a little bit. It is absolutely infuriating, if not impossible, to try dissection on plates that are wet.		#'''*[[User:Megan N McClean\|Megan N McClean]]''' When I can't age my dissection plates on my bench for a few days, I will stick them in the 30°C or 37°C warm room the morning of the day I dissect to dry them out a little bit. It is absolutely infuriating, if not impossible, to try dissection on plates that are wet.
-	~~Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.~~
	#'''*[[User:Megan N McClean\|Megan N McClean]]''' Different tetrad dissection protocols call for using different enzymes to digest the ascus wall. Our protocol uses a β-glucuronidase from Sigma (G7770) which is a mixture of enzymes derived from Helix pomatia (the Roman snail). Zymolyase, another commonly used enzyme, consists mostly of β-1,3-glucan laminaripentaohydrolase. It hydrolyzes glucose polymers at the β-1,3-glucan linkages releasing laminaripentaose as the principal product. β-glucuronidase catalyzes hydrolysis of β-D-glucuronic acid residues from the non-reducing end of mucopolysaccharides (also referred to as glycosaminoglycans).		#'''*[[User:Megan N McClean\|Megan N McClean]]''' Different tetrad dissection protocols call for using different enzymes to digest the ascus wall. Our protocol uses a β-glucuronidase from Sigma (G7770) which is a mixture of enzymes derived from Helix pomatia (the Roman snail). Zymolyase, another commonly used enzyme, consists mostly of β-1,3-glucan laminaripentaohydrolase. It hydrolyzes glucose polymers at the β-1,3-glucan linkages releasing laminaripentaose as the principal product. β-glucuronidase catalyzes hydrolysis of β-D-glucuronic acid residues from the non-reducing end of mucopolysaccharides (also referred to as glycosaminoglycans).

Megan N McClean: /* Dissection */

Megan N McClean — Fri, 30 Sep 2011 20:23:33 GMT

Dissection

←Older revision		Revision as of 20:23, 30 September 2011
Line 52:		Line 52:
	## Pick up the four spores with the microneedle. I like to place the needle very near to (but not touching) the spores, and if they are a genuine tetrad they will be sucked by surface tension as a group toward the needle.		## Pick up the four spores with the microneedle. I like to place the needle very near to (but not touching) the spores, and if they are a genuine tetrad they will be sucked by surface tension as a group toward the needle.
	## Place them on the agar in a click position at least 5 mm from the stripe of b-glucoronidase-treated cells. Note the position on the mechanical stage.		## Place them on the agar in a click position at least 5 mm from the stripe of b-glucoronidase-treated cells. Note the position on the mechanical stage.
-	*# To break apart the four spores, place the needle so that it barely touches the tetrad then gently tap the base of the microscope or table beside the microscope. This vibrates the needle enough to break apart the tetrad without jabbing the needle into the agar.	+	## To break apart the four spores, place the needle so that it barely touches the tetrad then gently tap the base of the microscope or table beside the microscope. This vibrates the needle enough to break apart the tetrad without jabbing the needle into the agar.
	## Pick up three spores. Move the stage 5 mm (one click) further away from the streak and deposit the three spores.		## Pick up three spores. Move the stage 5 mm (one click) further away from the streak and deposit the three spores.
	## Pick up two spores. Move the stage 5 mm (one click) away from the streak and deposit the two spores.		## Pick up two spores. Move the stage 5 mm (one click) away from the streak and deposit the two spores.

Megan N McClean: /* Dissection */

Megan N McClean — Fri, 30 Sep 2011 20:22:25 GMT

Dissection

←Older revision		Revision as of 20:22, 30 September 2011
Line 52:		Line 52:
	## Pick up the four spores with the microneedle. I like to place the needle very near to (but not touching) the spores, and if they are a genuine tetrad they will be sucked by surface tension as a group toward the needle.		## Pick up the four spores with the microneedle. I like to place the needle very near to (but not touching) the spores, and if they are a genuine tetrad they will be sucked by surface tension as a group toward the needle.
	## Place them on the agar in a click position at least 5 mm from the stripe of b-glucoronidase-treated cells. Note the position on the mechanical stage.		## Place them on the agar in a click position at least 5 mm from the stripe of b-glucoronidase-treated cells. Note the position on the mechanical stage.
-	## To break apart the four spores, place the needle so that it barely touches the tetrad then gently tap the base of the microscope or table beside the microscope. This vibrates the needle enough to break apart the tetrad without jabbing the needle into the agar.	+	*# To break apart the four spores, place the needle so that it barely touches the tetrad then gently tap the base of the microscope or table beside the microscope. This vibrates the needle enough to break apart the tetrad without jabbing the needle into the agar.
	## Pick up three spores. Move the stage 5 mm (one click) further away from the streak and deposit the three spores.		## Pick up three spores. Move the stage 5 mm (one click) further away from the streak and deposit the three spores.
	## Pick up two spores. Move the stage 5 mm (one click) away from the streak and deposit the two spores.		## Pick up two spores. Move the stage 5 mm (one click) away from the streak and deposit the two spores.

Megan N McClean: /* Appearance of Tetrads */

Megan N McClean — Fri, 30 Sep 2011 20:22:08 GMT

Appearance of Tetrads

←Older revision		Revision as of 20:22, 30 September 2011
Line 34:		Line 34:

	How to distinguish a tetrad from two budded cells adjacent to each other?		How to distinguish a tetrad from two budded cells adjacent to each other?
-	1) Generally, the spores of a tetrad are smaller than a budded cell.	+	# Generally, the spores of a tetrad are smaller than a budded cell.
-	2) Tetrads, if moved gently, will remain intact while two adjacent budded cells will not.	+	# Tetrads, if moved gently, will remain intact while two adjacent budded cells will not.
-	3) The four spores in a tetrad are often very similar in size, while a budded cell usually has a large mother cell and a smaller bud.	+	# The four spores in a tetrad are often very similar in size, while a budded cell usually has a large mother cell and a smaller bud.


		+	==Dissection==
		+	Dissecting scopes have a specially designed stage with “clicks” you can feel in both the x and y direction that allow even spacing of tetrads. The micromanipulator is used to control the position of the needle.

-	4 spores of a tetrad. After digestion, most tetrads should have this appearance. Tetrad with 3 spores visible in one focal plane and 4th spore visible in a second focal plane. Before digestion, many tetrads may have this appearance.	+	# Drop the needle to its lowest position by raising the arm of the micromanipulator (yes, this seems somewhat counterintuitive!!)
-	~~The Goal of your Dissection~~	+	# Move the turret of the scope so that there is no objective lens over the stage. This will give you room to position the plate.
-	~~Dissection~~	+	# Positioning your agar plate on the stage is like flying a helicopter, not a jet. Landing, come over high, then descend. Taking off, go straight up, then depart. The needle stands higher than the platform!
-	The dissecting scopes have a specially designed stage with “clicks” you can feel in both the x and y direction that allow even spacing of tetrads. The micromanipulator is used to control the position of the needle.	+	# Position the plate on the stage so that the two marks you made line up with the horizontal axis of the plate.
-		+	# Swing the objective into position, and move the stage so that the objective is centered over the swath where you dripped the digested sporulation down the plate.
-	1. Drop the needle to its lowest position by raising the arm of the micromanipulator (yes, this seems somewhat counterintuitive!!)	+	# Focus on the cells sitting on the surface of the plate (you will be focusing through the agar into the cells sitting on the surface).
-	2. Move the turret of the scope so that there is no objective lens over the stage. This will give you room to position the plate.	+	# Pick tetrads.
-	3. Positioning your agar plate on the stage is like flying a helicopter, not a jet. Landing, come over high, then descend. Taking off, go straight up, then depart. The needle stands higher than the platform!	+	## Move the microscope stage so that individual tetrads with a clear zone around them are visible. Raise the needle (by lowering the arm of the micromanipulator) until you can see it.
-	4. Position the plate on the stage so that the two marks you made line up with the horizontal axis of the plate.	+	## Pick up the four spores with the microneedle. I like to place the needle very near to (but not touching) the spores, and if they are a genuine tetrad they will be sucked by surface tension as a group toward the needle.
-	5. Swing the objective into position, and move the stage so that the objective is centered over the swath where you dripped the digested sporulation down the plate.	+	## Place them on the agar in a click position at least 5 mm from the stripe of b-glucoronidase-treated cells. Note the position on the mechanical stage.
-	6. Focus on the cells sitting on the surface of the plate (you will be focusing through the agar into the cells sitting on the surface).	+	## To break apart the four spores, place the needle so that it barely touches the tetrad then gently tap the base of the microscope or table beside the microscope. This vibrates the needle enough to break apart the tetrad without jabbing the needle into the agar.
-	7. Pick tetrads.	+	## Pick up three spores. Move the stage 5 mm (one click) further away from the streak and deposit the three spores.
-	i. Move the microscope stage so that individual tetrads with a clear zone around them are visible. Raise the needle (by lowering the arm of the micromanipulator) until you can see it.	+	## Pick up two spores. Move the stage 5 mm (one click) away from the streak and deposit the two spores.
-	~~ii.~~ Pick up the four spores with the microneedle. I like to place the needle very near to (but not touching) the spores, and if they are a genuine tetrad they will be sucked by surface tension as a group toward the needle.	+	## Pick up the remaining spore. Move the stage 5 mm (one click) and deposit the remaining spore.
-	~~iii.~~ Place them on the agar in a click position at least 5 mm from the stripe of b-glucoronidase-treated cells. Note the position on the mechanical stage.	+	## Move the microscope stage to the left or right 5 mm (one click) from the line of the four spores, and select another four-spore cluster. Separate the spores as before by 5-mm intervals. By this method, 10 tetrads can be dissected on each side of the YPD plate.
-	~~iv.~~ To break apart the four spores, place the needle so that it barely touches the tetrad then gently tap the base of the microscope or table beside the microscope. This vibrates the needle enough to break apart the tetrad without jabbing the needle into the agar.	+	## Remove the YPD plate from the stage, taking care not to break the microneedle.
-	v. Pick up three spores. Move the stage 5 mm (one click) further away from the streak and deposit the three spores.	+	# Incubate the plate for 2-3 days at room temperature.
-	~~vi.~~ Pick up two spores. Move the stage 5 mm (one click) away from the streak and deposit the two spores.	+
-	~~vii.~~ Pick up the remaining spore. Move the stage 5 mm (one click) and deposit the remaining spore.	+
-	~~viii.~~ Move the microscope stage to the left or right 5 mm (one click) from the line of the four spores, and select another four-spore cluster. Separate the spores as before by 5-mm intervals. By this method, 10 tetrads can be dissected on each side of the YPD plate.	+
-	~~ix.~~ Remove the YPD plate from the stage, taking care not to break the microneedle.	+
-	8. Incubate the plate for 2-3 days at room temperature~~. We will test the phenotype (auxotrophic or not) and mating type of your spores~~.	+

	==Notes==		==Notes==

Megan N McClean: /* Digestion */

Megan N McClean — Fri, 30 Sep 2011 20:20:14 GMT

Digestion

←Older revision		Revision as of 20:20, 30 September 2011
Line 17:		Line 17:
	==Protocol==		==Protocol==
	===Digestion===		===Digestion===
-	The spores are contained in a specialized cell wall called an ascus. In order to be able to separate the spores, the ascus is digested with the enzyme B-glucuronidase. Ideally, the ascus is digested just enough that the spores can be separated but not so much that the spores fall apart entirely.	+	The spores are contained in a specialized cell wall called an ascus. In order to be able to separate the spores, the ascus is digested with the enzyme β-glucuronidase. Ideally, the ascus is digested just enough that the spores can be separated but not so much that the spores fall apart entirely.

-	# Spin down 250 ul of sporulated culture.	+	# Spin down 250 μL of sporulated culture.
-	# Resuspend in 250 ul sterile water.	+	# Resuspend in 250 μL sterile water.
-	# Mix 17 ul washed culture with 3 ~~ul B~~-glucuronidase (can vary by strain). Set up three tubes like this – you will stop them at different time points to check digestion.	+	# Mix 17 μL washed culture with 3 μL β-glucuronidase (can vary by strain). Set up three tubes like this – you will stop them at different time points to check digestion.
-	# Let sit at room temperature 20, 30, and 40 minutes. Stop the digestions as in step 5. (The ideal timing for digestion varies by strain, culture, and ambient conditions – if you are having trouble with your dissections, you may need to try less or more time. ~~Check with an instructor for their opinion if you are having trouble with your dissection.)~~	+	# Let sit at room temperature 20, 30, and 40 minutes. Stop the digestions as in step 5. (The ideal timing for digestion varies by strain, culture, and ambient conditions – if you are having trouble with your dissections, you may need to try less or more time.)
-	# Gently add 100 ul water. The goal is to suspend the cells without breaking up the tetrads. Tap the tube gently to mix.	+	# Gently add 100 μL water. The goal is to suspend the cells without breaking up the tetrads. Tap the tube gently to mix.
	# Transfer 5 μl of each test digestion to a slide and cover each with individual coverslips. Check for digestion under the upright microscope (see next section on appearance of tetrads).		# Transfer 5 μl of each test digestion to a slide and cover each with individual coverslips. Check for digestion under the upright microscope (see next section on appearance of tetrads).
	# Mark the plate at opposite edges to indicate the center of the plate (see following diagram).		# Mark the plate at opposite edges to indicate the center of the plate (see following diagram).

Megan N McClean: /* Stock Solutions */

Megan N McClean — Fri, 30 Sep 2011 20:11:40 GMT

Stock Solutions

←Older revision		Revision as of 20:11, 30 September 2011
Line 13:		Line 13:
	**Everyone has a particular way that they like their plates for tetrad dissection. You are basically aiming for ''dry'' and ''level''. To make dissection plates, add 25 ml YPD with a plastic strippette to plates on a very level surface. Once solid, invert. Let dry at room temperature for ~3 days. Bag. These plates are best after aging for a while.		**Everyone has a particular way that they like their plates for tetrad dissection. You are basically aiming for ''dry'' and ''level''. To make dissection plates, add 25 ml YPD with a plastic strippette to plates on a very level surface. Once solid, invert. Let dry at room temperature for ~3 days. Bag. These plates are best after aging for a while.

-	~~==Stock Solutions==~~

-	~~'''Stock Solution 1'''~~
-	* This is a very simple solution, so we only need a one line description of how to make it.
-
-	~~'''Stock Solution 2'''~~
-
-	~~This is a more involved solution, so we will describe how to make it in several steps:~~
-	~~# Step 1~~
-	~~# Step 2~~
-	~~# Step 3~~

	==Protocol==		==Protocol==

Megan N McClean: /* Protocol */

Megan N McClean — Fri, 30 Sep 2011 18:56:35 GMT

Protocol

←Older revision		Revision as of 18:56, 30 September 2011
Line 26:		Line 26:

	==Protocol==		==Protocol==
-	~~# Step 1~~	+	===Digestion===
-	~~# Step 2~~	+	The spores are contained in a specialized cell wall called an ascus. In order to be able to separate the spores, the ascus is digested with the enzyme B-glucuronidase. Ideally, the ascus is digested just enough that the spores can be separated but not so much that the spores fall apart entirely.
-	~~# Step 3~~	+

		+	# Spin down 250 ul of sporulated culture.
		+	# Resuspend in 250 ul sterile water.
		+	# Mix 17 ul washed culture with 3 ul B-glucuronidase (can vary by strain). Set up three tubes like this – you will stop them at different time points to check digestion.
		+	# Let sit at room temperature 20, 30, and 40 minutes. Stop the digestions as in step 5. (The ideal timing for digestion varies by strain, culture, and ambient conditions – if you are having trouble with your dissections, you may need to try less or more time. Check with an instructor for their opinion if you are having trouble with your dissection.)
		+	# Gently add 100 ul water. The goal is to suspend the cells without breaking up the tetrads. Tap the tube gently to mix.
		+	# Transfer 5 μl of each test digestion to a slide and cover each with individual coverslips. Check for digestion under the upright microscope (see next section on appearance of tetrads).
		+	# Mark the plate at opposite edges to indicate the center of the plate (see following diagram).
		+	# Resuspend the digested cells from your chosen timepoint. by gently tapping or pipetting (they will probably have settled while you were looking at the digestions under the microscope). Drip 20 μl down the imaginary center line you just indicated, beginning from one edge.
		+	# Let the digested culture fully absorb into the plate.
		+	# Dissect (see dissection section). If you dissect on a different day, you must begin with a fresh plate and set up a new dissection. The sporulation cultures are good for weeks, though the spore viability may start to decrease.
		+
		+	===Appearance of Tetrads===
		+	Before digestion, tetrads are held tightly in a tetrahedron and it is often difficult ot see all four spores at once. After digestion they relax into a diamond shape.
		+
		+	How to distinguish a tetrad from two budded cells adjacent to each other?
		+	1) Generally, the spores of a tetrad are smaller than a budded cell.
		+	2) Tetrads, if moved gently, will remain intact while two adjacent budded cells will not.
		+	3) The four spores in a tetrad are often very similar in size, while a budded cell usually has a large mother cell and a smaller bud.
		+
		+
		+
		+	4 spores of a tetrad. After digestion, most tetrads should have this appearance. Tetrad with 3 spores visible in one focal plane and 4th spore visible in a second focal plane. Before digestion, many tetrads may have this appearance.
		+	The Goal of your Dissection
		+	Dissection
		+	The dissecting scopes have a specially designed stage with “clicks” you can feel in both the x and y direction that allow even spacing of tetrads. The micromanipulator is used to control the position of the needle.
		+
		+	1. Drop the needle to its lowest position by raising the arm of the micromanipulator (yes, this seems somewhat counterintuitive!!)
		+	2. Move the turret of the scope so that there is no objective lens over the stage. This will give you room to position the plate.
		+	3. Positioning your agar plate on the stage is like flying a helicopter, not a jet. Landing, come over high, then descend. Taking off, go straight up, then depart. The needle stands higher than the platform!
		+	4. Position the plate on the stage so that the two marks you made line up with the horizontal axis of the plate.
		+	5. Swing the objective into position, and move the stage so that the objective is centered over the swath where you dripped the digested sporulation down the plate.
		+	6. Focus on the cells sitting on the surface of the plate (you will be focusing through the agar into the cells sitting on the surface).
		+	7. Pick tetrads.
		+	i. Move the microscope stage so that individual tetrads with a clear zone around them are visible. Raise the needle (by lowering the arm of the micromanipulator) until you can see it.
		+	ii. Pick up the four spores with the microneedle. I like to place the needle very near to (but not touching) the spores, and if they are a genuine tetrad they will be sucked by surface tension as a group toward the needle.
		+	iii. Place them on the agar in a click position at least 5 mm from the stripe of b-glucoronidase-treated cells. Note the position on the mechanical stage.
		+	iv. To break apart the four spores, place the needle so that it barely touches the tetrad then gently tap the base of the microscope or table beside the microscope. This vibrates the needle enough to break apart the tetrad without jabbing the needle into the agar.
		+	v. Pick up three spores. Move the stage 5 mm (one click) further away from the streak and deposit the three spores.
		+	vi. Pick up two spores. Move the stage 5 mm (one click) away from the streak and deposit the two spores.
		+	vii. Pick up the remaining spore. Move the stage 5 mm (one click) and deposit the remaining spore.
		+	viii. Move the microscope stage to the left or right 5 mm (one click) from the line of the four spores, and select another four-spore cluster. Separate the spores as before by 5-mm intervals. By this method, 10 tetrads can be dissected on each side of the YPD plate.
		+	ix. Remove the YPD plate from the stage, taking care not to break the microneedle.
		+	8. Incubate the plate for 2-3 days at room temperature. We will test the phenotype (auxotrophic or not) and mating type of your spores.

	==Notes==		==Notes==