Haynes:UPLassay - Revision history

Karmella Haynes: /* Universal Probe Library Assay */

Karmella Haynes — Mon, 25 Feb 2013 18:43:34 GMT

Universal Probe Library Assay

←Older revision		Revision as of 18:43, 25 February 2013
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	Variation 1<br> [[Image:Haynes_UPL_fig1.png\|300px\|Figure 1]] <br>		Variation 1<br> [[Image:Haynes_UPL_fig1.png\|300px\|Figure 1]] <br>
	Variation 2<br> [[Image:Haynes_UPL_fig2.png\|300px\|Figure 1]]<br>		Variation 2<br> [[Image:Haynes_UPL_fig2.png\|300px\|Figure 1]]<br>
-	Both Variations (1 and 2) are correct. Choose a format that helps you to easily keep track of the samples. If you have a large experiment, try to fit as many reactions on the ~~well~~ as you can (to avoid wasting plates), but also keep the samples arranged in an orderly fashion so that the set-up won't confuse you.	+	Both Variations (1 and 2) are correct. Choose a format that helps you to easily keep track of the samples. If you have a large experiment, try to fit as many reactions on the plate as you can (to avoid wasting plates), but also keep the samples arranged in an orderly fashion so that the set-up won't confuse you.
	\|}		\|}

Karmella Haynes: /* Universal Probe Library Assay */

Karmella Haynes — Wed, 06 Feb 2013 03:40:03 GMT

Universal Probe Library Assay

←Older revision		Revision as of 03:40, 6 February 2013
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	\|}		\|}
	\| '''PLATE LAYOUT'''<br>		\| '''PLATE LAYOUT'''<br>
-	Both of these are correct. Use a format that helps you to easily keep track of the samples. If you have a large experiment, try to fit as many reactions on the well as you can (to avoid wasting plates), but also keep the samples arranged in an orderly fashion so that the set-up won't confuse you.
	Variation 1<br> [[Image:Haynes_UPL_fig1.png\|300px\|Figure 1]] <br>		Variation 1<br> [[Image:Haynes_UPL_fig1.png\|300px\|Figure 1]] <br>
-	Variation 2<br> [[Image:Haynes_UPL_fig2.png\|300px\|Figure 1]]	+	Variation 2<br> [[Image:Haynes_UPL_fig2.png\|300px\|Figure 1]]<br>
		+	Both Variations (1 and 2) are correct. Choose a format that helps you to easily keep track of the samples. If you have a large experiment, try to fit as many reactions on the well as you can (to avoid wasting plates), but also keep the samples arranged in an orderly fashion so that the set-up won't confuse you.
	\|}		\|}

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	5. '''Reaction set-up: loading the 96-well plate'''<br>		5. '''Reaction set-up: loading the 96-well plate'''<br>
-	The following ~~illustration uses~~ Variation 2	+	The following illustrations use Plate Layout Variation 2...

	{\| width=800px		{\| width=800px

Karmella Haynes: /* Universal Probe Library Assay */

Karmella Haynes — Wed, 06 Feb 2013 03:38:37 GMT

Universal Probe Library Assay

←Older revision		Revision as of 03:38, 6 February 2013
Line 72:		Line 72:
	\|}		\|}
	\| '''PLATE LAYOUT'''<br>		\| '''PLATE LAYOUT'''<br>
		+	Both of these are correct. Use a format that helps you to easily keep track of the samples. If you have a large experiment, try to fit as many reactions on the well as you can (to avoid wasting plates), but also keep the samples arranged in an orderly fashion so that the set-up won't confuse you.
	Variation 1<br> [[Image:Haynes_UPL_fig1.png\|300px\|Figure 1]] <br>		Variation 1<br> [[Image:Haynes_UPL_fig1.png\|300px\|Figure 1]] <br>
	Variation 2<br> [[Image:Haynes_UPL_fig2.png\|300px\|Figure 1]]		Variation 2<br> [[Image:Haynes_UPL_fig2.png\|300px\|Figure 1]]
Line 148:		Line 149:

	5. '''Reaction set-up: loading the 96-well plate'''<br>		5. '''Reaction set-up: loading the 96-well plate'''<br>
		+	The following illustration uses Variation 2

	{\| width=800px		{\| width=800px

Karmella Haynes: /* Universal Probe Library Assay */

Karmella Haynes — Sun, 20 Jan 2013 16:26:25 GMT

Universal Probe Library Assay

←Older revision		Revision as of 16:26, 20 January 2013
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	** Gene targets + GAPD reference = 5		** Gene targets + GAPD reference = 5
	** Replicates per reaction = 3		** Replicates per reaction = 3
-	** '''Wells ~~need~~ = 3 * 5 * 3 = 45'''	+	** '''Wells needed = 3 * 5 * 3 = 45'''

Karmella Haynes: /* Universal Probe Library Assay */

Karmella Haynes — Sun, 20 Jan 2013 16:25:02 GMT

Universal Probe Library Assay

←Older revision		Revision as of 16:25, 20 January 2013
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	1. '''Design your primers'''<br>		1. '''Design your primers'''<br>
-	* Each gene you analyze requires a ~~three-part oligo~~ set: '''forward primer''', '''reverse primer''', and a '''UPL probe'''.	+	* Each gene you analyze requires a set of three oligos: '''forward primer''', '''reverse primer''', and a '''UPL probe'''.
	* Use Roche's [http://www.roche-applied-science.com/sis/rtpcr/upl/index.jsp?id=UP030000 Assay Design Center] to design optimal primers, and to identify the right UPL probe for your gene(s) of interest.		* Use Roche's [http://www.roche-applied-science.com/sis/rtpcr/upl/index.jsp?id=UP030000 Assay Design Center] to design optimal primers, and to identify the right UPL probe for your gene(s) of interest.
	* The forward and reverse primers need to be ordered from a DNA synthesis company (e.g., IDT DNA, Promega, etc.), and the UPL oligo comes from Roche.		* The forward and reverse primers need to be ordered from a DNA synthesis company (e.g., IDT DNA, Promega, etc.), and the UPL oligo comes from Roche.

Karmella Haynes: /* Universal Probe Library Assay */

Karmella Haynes — Sun, 20 Jan 2013 16:24:35 GMT

Universal Probe Library Assay

←Older revision		Revision as of 16:24, 20 January 2013
Line 18:		Line 18:

	1. '''Design your primers'''<br>		1. '''Design your primers'''<br>
-	* Each gene you analyze requires a three-part ~~primer~~ set: '''forward primer''', '''reverse primer''', and a '''UPL probe'''.	+	* Each gene you analyze requires a three-part oligo set: '''forward primer''', '''reverse primer''', and a '''UPL probe'''.
	* Use Roche's [http://www.roche-applied-science.com/sis/rtpcr/upl/index.jsp?id=UP030000 Assay Design Center] to design optimal primers, and to identify the right UPL probe for your gene(s) of interest.		* Use Roche's [http://www.roche-applied-science.com/sis/rtpcr/upl/index.jsp?id=UP030000 Assay Design Center] to design optimal primers, and to identify the right UPL probe for your gene(s) of interest.
	* The forward and reverse primers need to be ordered from a DNA synthesis company (e.g., IDT DNA, Promega, etc.), and the UPL oligo comes from Roche.		* The forward and reverse primers need to be ordered from a DNA synthesis company (e.g., IDT DNA, Promega, etc.), and the UPL oligo comes from Roche.

Karmella Haynes: /* Universal Probe Library Assay */

Karmella Haynes — Sun, 20 Jan 2013 16:24:13 GMT

Universal Probe Library Assay

←Older revision		Revision as of 16:24, 20 January 2013
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	* Make a 1:10 dilution of cDNA by adding 10 μL of the stock cDNA to 90 μL of PCR H<sub>2</sub>O.		* Make a 1:10 dilution of cDNA by adding 10 μL of the stock cDNA to 90 μL of PCR H<sub>2</sub>O.
	* Make enough Template master mix for your plate...		* Make enough Template master mix for your plate...
-	** '''Treated ~~cell~~ cDNA''' is in Reactions 1, 2, 3, 4 and 5 = 5	+	** '''Treated cells cDNA''' is in Reactions 1, 2, 3, 4 and 5 = 5
	** Replicates per reaction = 3		** Replicates per reaction = 3
	** '''Master mix amount = 5 * 3 + 1 (to allow for pipetting error) = 16'''		** '''Master mix amount = 5 * 3 + 1 (to allow for pipetting error) = 16'''
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	Resulting 1.5 mL tubes:		Resulting 1.5 mL tubes:
-	* '''T1''' - ~~untreated~~ cDNA, 84.5 μL	+	* '''T1''' - treated cells cDNA, 84.5 μL
-	* '''T2''' - ~~treated~~ cDNA, 84.5 μL	+	* '''T2''' - untreated cells cDNA, 84.5 μL
-	* '''T3''' - no template, 84.5 μL	+	* '''T3''' - no template control, 84.5 μL

Karmella Haynes: /* Universal Probe Library Assay */

Karmella Haynes — Sun, 20 Jan 2013 16:20:24 GMT

Universal Probe Library Assay

←Older revision		Revision as of 16:20, 20 January 2013
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	\|}		\|}

-	* A single plate contains 96 wells ~~(as shown below)~~. To insure accuracy, '''three technical replicates per reaction''' (Rxn) are required	+	* A single plate contains 96 wells. To insure accuracy, '''three technical replicates per reaction''' (Rxn) are required
	* If you need more than 96 wells, you must split the experiment over multiple plates.		* If you need more than 96 wells, you must split the experiment over multiple plates.
	* It is absolutely critical that you keep a '''reaction list''' and '''plate layout''' in your notes. Your plate set-up will probably vary for each run.		* It is absolutely critical that you keep a '''reaction list''' and '''plate layout''' in your notes. Your plate set-up will probably vary for each run.

Karmella Haynes: /* Universal Probe Library Assay */

Karmella Haynes — Sun, 20 Jan 2013 04:34:10 GMT

Universal Probe Library Assay

←Older revision		Revision as of 04:34, 20 January 2013
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	\|- valign="top"		\|- valign="top"
	\| Transfer 15 μL of solution from A1 into A2, and A3. Use a fresh pipette tip to do the same for A4-6, and A7-8.		\| Transfer 15 μL of solution from A1 into A2, and A3. Use a fresh pipette tip to do the same for A4-6, and A7-8.
-	\| align="right" \| [[Image:Haynes_UPL_fig5.png\|~~350px~~\|Figure 5]]	+	\| align="right" \| [[Image:Haynes_UPL_fig5.png\|300px\|Figure 5]]
	\|- valign="top"		\|- valign="top"
	\| Repeat this procedure for the rest of the plate		\| Repeat this procedure for the rest of the plate
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	\|}		\|}

-	The plate is now ready to load into the Light Cycler 480!	+	* Seal the plate with clear film.
		+	* The plate is now ready to load into the Light Cycler 480!

Karmella Haynes: /* Universal Probe Library Assay */

Karmella Haynes — Sun, 20 Jan 2013 04:32:01 GMT

Universal Probe Library Assay

←Older revision		Revision as of 04:32, 20 January 2013
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	\|- valign="top"		\|- valign="top"
	\| Pipette 19.5 μL of cDNA Template master mixes into the first well of each 3-well group<br>		\| Pipette 19.5 μL of cDNA Template master mixes into the first well of each 3-well group<br>
-	\| ~~halign~~="right" \| [[Image:Haynes_UPL_fig3.png\|300px\|Figure 3]]	+	\| align="right" \| [[Image:Haynes_UPL_fig3.png\|300px\|Figure 3]]
	\|- valign="top"		\|- valign="top"
-	\| Add 25.5 μL of Gene Target master mix to the cDNA. After each addition, mix by gently pipetting up and down 3 - 5 times (without making bubbles). \|\| [[Image:Haynes_UPL_fig4.png\|350px\|Figure 4]]	+	\| Add 25.5 μL of Gene Target master mix to the cDNA. After each addition, mix by gently pipetting up and down 3 - 5 times (without making bubbles).
		+	\| align="right" \| [[Image:Haynes_UPL_fig4.png\|350px\|Figure 4]]
	\|- valign="top"		\|- valign="top"
-	\| Transfer 15 μL of solution from A1 into A2, and A3. Use a fresh pipette tip to do the same for A4-6, and A7-8. \|\| [[Image:Haynes_UPL_fig5.png\|350px\|Figure 5]]	+	\| Transfer 15 μL of solution from A1 into A2, and A3. Use a fresh pipette tip to do the same for A4-6, and A7-8.
		+	\| align="right" \| [[Image:Haynes_UPL_fig5.png\|350px\|Figure 5]]
	\|- valign="top"		\|- valign="top"
-	\| Repeat this procedure for the rest of the plate \|\| [[Image:Haynes_UPL_fig2.png\|350px\|Figure 2]]	+	\| Repeat this procedure for the rest of the plate
		+	\| align="right" \| [[Image:Haynes_UPL_fig2.png\|350px\|Figure 2]]
	\|}		\|}

	The plate is now ready to load into the Light Cycler 480!		The plate is now ready to load into the Light Cycler 480!