BME103:T130 Group 13 l2 - Revision history

Emily Herring: /* Protocols */

Emily Herring — Thu, 29 Nov 2012 20:26:55 GMT

Protocols

←Older revision		Revision as of 20:26, 29 November 2012
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	After assembling the fluorimeter, you can now determine if you've amplified the targeted DNA in your PCR experiment. Using the Fluorimeter and the smartphone app, you can calculate the relative amount of DNA through fluorescence, which is generated by excitation and emission wavelengths. In order to detect fluorescence when dsDNA is present, you'll be using SYBR Green I because it's more safer compared to other dyes. With that being said, gloves must be worn when handling any liquid containing SYBR Green I. The fluorimeter itself is a very simple machine because it uses optical caustic, a special type of optics that completely removes the need for lasers, mirrors, or lenses. Also the flourimeter is battery-powered, lightweight and portable; this allows every student to have one of these at their lab table. Following the steps below, you can easily learn how to dye your amplified DNA. <br>		After assembling the fluorimeter, you can now determine if you've amplified the targeted DNA in your PCR experiment. Using the Fluorimeter and the smartphone app, you can calculate the relative amount of DNA through fluorescence, which is generated by excitation and emission wavelengths. In order to detect fluorescence when dsDNA is present, you'll be using SYBR Green I because it's more safer compared to other dyes. With that being said, gloves must be worn when handling any liquid containing SYBR Green I. The fluorimeter itself is a very simple machine because it uses optical caustic, a special type of optics that completely removes the need for lasers, mirrors, or lenses. Also the flourimeter is battery-powered, lightweight and portable; this allows every student to have one of these at their lab table. Following the steps below, you can easily learn how to dye your amplified DNA. <br>
		+
	1)On your lab table, you'll find eight samples from the Open PCR, 1 DNA sample(calf thymus standard at 2 micrograms/mL), and water from the scintillation vial (white cap) to analyze. <br>		1)On your lab table, you'll find eight samples from the Open PCR, 1 DNA sample(calf thymus standard at 2 micrograms/mL), and water from the scintillation vial (white cap) to analyze. <br>
	2)With a permanent marker, label your Eppendorf tubes and number your pipettes (on the bulb part) so that no cross-contamination will occurs. At the end, you should have 10 Eppendorf tubes and 10 pipettes clearly labeled (see Table 3). REMINDER: Use only 1 transfer pipette per sample!!! <br>		2)With a permanent marker, label your Eppendorf tubes and number your pipettes (on the bulb part) so that no cross-contamination will occurs. At the end, you should have 10 Eppendorf tubes and 10 pipettes clearly labeled (see Table 3). REMINDER: Use only 1 transfer pipette per sample!!! <br>

Emily Herring: /* Protocols */

Emily Herring — Thu, 29 Nov 2012 20:26:22 GMT

Protocols

←Older revision		Revision as of 20:26, 29 November 2012
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	12) Repeat steps 5-10 four more times in 5 different positions. <br>		12) Repeat steps 5-10 four more times in 5 different positions. <br>

-	After assembling the fluorimeter, you can now determine if you've amplified the targeted DNA in your PCR experiment. Using the Fluorimeter and the smartphone app, you can calculate the relative amount of DNA through fluorescence, which is generated by excitation and emission wavelengths. In order to detect fluorescence when dsDNA is present, you'll be using SYBR Green I because it's more safer compared to other dyes. With that being said, gloves must be worn when handling any liquid containing SYBR Green I. The fluorimeter itself is a very simple machine because it uses optical caustic, a special type of optics that completely removes the need for lasers, mirrors, or lenses. Also the flourimeter is battery-powered, lightweight and portable; this allows every student to have one of these at their lab table. Following the steps below, you can easily learn how to dye your amplified DNA.	+	After assembling the fluorimeter, you can now determine if you've amplified the targeted DNA in your PCR experiment. Using the Fluorimeter and the smartphone app, you can calculate the relative amount of DNA through fluorescence, which is generated by excitation and emission wavelengths. In order to detect fluorescence when dsDNA is present, you'll be using SYBR Green I because it's more safer compared to other dyes. With that being said, gloves must be worn when handling any liquid containing SYBR Green I. The fluorimeter itself is a very simple machine because it uses optical caustic, a special type of optics that completely removes the need for lasers, mirrors, or lenses. Also the flourimeter is battery-powered, lightweight and portable; this allows every student to have one of these at their lab table. Following the steps below, you can easily learn how to dye your amplified DNA. <br>
	1)On your lab table, you'll find eight samples from the Open PCR, 1 DNA sample(calf thymus standard at 2 micrograms/mL), and water from the scintillation vial (white cap) to analyze. <br>		1)On your lab table, you'll find eight samples from the Open PCR, 1 DNA sample(calf thymus standard at 2 micrograms/mL), and water from the scintillation vial (white cap) to analyze. <br>
	2)With a permanent marker, label your Eppendorf tubes and number your pipettes (on the bulb part) so that no cross-contamination will occurs. At the end, you should have 10 Eppendorf tubes and 10 pipettes clearly labeled (see Table 3). REMINDER: Use only 1 transfer pipette per sample!!! <br>		2)With a permanent marker, label your Eppendorf tubes and number your pipettes (on the bulb part) so that no cross-contamination will occurs. At the end, you should have 10 Eppendorf tubes and 10 pipettes clearly labeled (see Table 3). REMINDER: Use only 1 transfer pipette per sample!!! <br>

Sudarshan Iyer: /* Thermal Cycler Engineering */

Sudarshan Iyer — Thu, 29 Nov 2012 19:03:30 GMT

Thermal Cycler Engineering

←Older revision		Revision as of 19:03, 29 November 2012
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	'''System Design'''<br>		'''System Design'''<br>
-	[[Image:BME103_13_130_HingedLid_3.JPG\|600px\|thumb\|A-Absent Heated Lid Handle\|thumb\|A-Absent Heating Lid Handle <br> B-~~Looser~~ Latch\|left]] <br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>	+	[[Image:BME103_13_130_HingedLid_3.JPG\|600px\|thumb\|A-Absent Heated Lid Handle\|thumb\|A-Absent Heating Lid Handle <br> B-Loose Latch\|left]] <br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>



	'''Key Features'''<br>		'''Key Features'''<br>
-	The new design focuses on making the Open PCR easier to use. To accomplish this goal based on the design, the new Open PCR design ~~has omitted its~~ Heated Lid Handle by making the Heated Lid set in a certain position based on the national standards of the testing vials. Thus, ~~decreasing~~ the error in heating based on a poorly adjusted lid because of this design. The new design also focuses on an improved latching system. This latch previously had difficulties opening and closing because of the tight fit. This has been ameliorated by making the latch ~~looser~~ in order to open and close more easily.	+	The new design focuses on making the Open PCR easier to use. To accomplish this goal based on the design, the new Open PCR design omits the Heated Lid Handle on the old design by making the Heated Lid set in a certain position based on the national standards of the testing vials. Thus, this decreases the error in heating based on a poorly adjusted lid because of this design. The new design also focuses on an improved latching system. This latch previously had difficulties opening and closing because of the tight fit. This has been ameliorated by making the latch loose in order to open and close more easily.


	'''Instructions'''<br>		'''Instructions'''<br>
-	To access the 16 tube rack, gently pull up on the lip of the lid (located on the same side as the latch). After loading or unloading the tube rack, close the lid by slowly, yet firmly pushing down on the top of the lid until it clicks into place. Make sure to double check to see if it ~~closed~~ all the way by slightly pulling up on the lid without opening it, to see if the latch prevents it from opening. Now you are free to run the test. The rest of the machine should work exactly as it did before the moderation.	+	To access the 16 tube rack, gently pull up on the lip of the lid (located on the same side as the latch). After loading or unloading the tube rack, close the lid by slowly, yet firmly pushing down on the top of the lid until it clicks into place. Make sure to double check to see if it closes all the way by slightly pulling up on the lid without opening it, to see if the latch prevents it from opening. Now you are free to run the test. The rest of the machine should work exactly as it did before the moderation.

	<!--- From Week 4 exercise --->		<!--- From Week 4 exercise --->

Ujwala Vaka: /* OUR TEAM */

Ujwala Vaka — Thu, 29 Nov 2012 18:55:30 GMT

OUR TEAM

←Older revision		Revision as of 18:55, 29 November 2012
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	\| [[Image:Voltage_main.jpg\|100px\|thumb\|Name: Garrett Repp<br>Open PCR machine engineer]]		\| [[Image:Voltage_main.jpg\|100px\|thumb\|Name: Garrett Repp<br>Open PCR machine engineer]]
	\| [[Image:Joseph_pic.jpeg\|100px\|thumb\|Name: Joseph Rosario<br>Open PCR machine engineer]]		\| [[Image:Joseph_pic.jpeg\|100px\|thumb\|Name: Joseph Rosario<br>Open PCR machine engineer]]
-	\| [[Image:~~BME103student~~.jpg\|100px\|thumb\|Name: Ujwala Vaka<br>Experimental protocol planner]]	+	\| [[Image:dnaproblems.jpg\|100px\|100px\|thumb\|Name: Ujwala Vaka<br>Experimental protocol planner]]
	\| [[Image:BME103_Group13_emily.jpg\|100px\|thumb\|Name: Emily Herring<br>Experimental protocol planner]]		\| [[Image:BME103_Group13_emily.jpg\|100px\|thumb\|Name: Emily Herring<br>Experimental protocol planner]]
	\| [[Image:BME103_Group13_SIavatar.jpg\|100px\|thumb\|Name: Sudarshan Iyer<br>Research and Development scientist]]		\| [[Image:BME103_Group13_SIavatar.jpg\|100px\|thumb\|Name: Sudarshan Iyer<br>Research and Development scientist]]

Ujwala Vaka: /* Protocols */

Ujwala Vaka — Thu, 29 Nov 2012 18:54:44 GMT

Protocols

←Older revision		Revision as of 18:54, 29 November 2012
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	{\| border="1" cellpadding="5" cellspacing="0" align="center"		{\| border="1" cellpadding="5" cellspacing="0" align="center"
	! scope="col" \| Supplied in Kit		! scope="col" \| Supplied in Kit
-	~~! scope="col" \| Amount~~

	\|-		\|-
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	{\| border="1" cellpadding="5" cellspacing="0" align="center"		{\| border="1" cellpadding="5" cellspacing="0" align="center"
	! scope="col" \| Supplied by User		! scope="col" \| Supplied by User
-	~~! scope="col" \| Amount~~

	\|-		\|-

Ujwala Vaka: /* Protocols */

Ujwala Vaka — Thu, 29 Nov 2012 18:53:56 GMT

Protocols

←Older revision		Revision as of 18:53, 29 November 2012
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	\|-		\|-
	\|PCR Master Mix		\|PCR Master Mix
-	~~\|20 containers~~

	\|-		\|-
	\|PCR Machine		\|PCR Machine
-	\|1

	\|-		\|-
	\|SYBR Green I		\|SYBR Green I
-	\|

	\|-		\|-
	\|Calf Thymus DNA Solution		\|Calf Thymus DNA Solution
-	\|

	\|-		\|-
	\|Fluorimeter Kit		\|Fluorimeter Kit
-	\|1

	\|-		\|-
	\|Mat with measurements		\|Mat with measurements
-	\|1
	\|}		\|}

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	\|-		\|-
	\|Smartphone		\|Smartphone
-	\|1

	\|-		\|-
	\|Pipettes		\|Pipettes
-	~~\|12~~

	\|-		\|-
	\|Eppendorf Tubes		\|Eppendorf Tubes
-	\|8

	\|-		\|-
	\|DNA Samples		\|DNA Samples
-	\|

	\|-		\|-
	\|Water		\|Water
-	\|
	\|}		\|}

Sudarshan Iyer: /* Research and Development */

Sudarshan Iyer — Thu, 29 Nov 2012 07:17:31 GMT

Research and Development

←Older revision		Revision as of 07:17, 29 November 2012
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	'''Illustration'''		'''Illustration'''
	<br><br>[[Image:BME103_Group13_SIPrimer.png]]		<br><br>[[Image:BME103_Group13_SIPrimer.png]]
		+
		+	'''Bayes' Rule: Test of Reliability'''
		+	<br><br>A nucleic acid test is only as useful as its level of reliability. If the test is not reliable and used, people who have a mutated T will be told they have or will have hipohydrotic ectodermal dysplasia when they actually do not. Bayes' Rule provides an equation that can be used to determine the probability that a person who has a mutated T will have hipohydrotic ectodermal dysplasia. The equation with the necessary variables is displayed below.
		+	<br>[[Image:BME103_Group13_SIBRule.png]]

	<!--- Include an illustration that shows how your system's primers allow specific amplification of the disease-related SNP --->		<!--- Include an illustration that shows how your system's primers allow specific amplification of the disease-related SNP --->

Sudarshan Iyer: /* Research and Development */

Sudarshan Iyer — Thu, 29 Nov 2012 07:07:27 GMT

Research and Development

←Older revision		Revision as of 07:07, 29 November 2012
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	'''Illustration'''		'''Illustration'''
		+	<br><br>[[Image:BME103_Group13_SIPrimer.png]]

	<!--- Include an illustration that shows how your system's primers allow specific amplification of the disease-related SNP --->		<!--- Include an illustration that shows how your system's primers allow specific amplification of the disease-related SNP --->

Sudarshan Iyer: /* Research and Development */

Sudarshan Iyer — Thu, 29 Nov 2012 07:06:16 GMT

Research and Development

←Older revision		Revision as of 07:06, 29 November 2012
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	'''Primer Design'''		'''Primer Design'''
	<br><br>Since this SNP lies on the forward strand, the primer used to facilitate a DNA signal of the mutated DNA will include a forward primer containing the mutation and a reverse primer 200 base pairs to the right of the mutation, with each primer being 20 base pairs long.		<br><br>Since this SNP lies on the forward strand, the primer used to facilitate a DNA signal of the mutated DNA will include a forward primer containing the mutation and a reverse primer 200 base pairs to the right of the mutation, with each primer being 20 base pairs long.
-	<br>Forward Primer (5' to 3'): [[Image:BME103_Group13_SIForwardprimer.png]]	+	<br><br>Forward Primer (5' to 3'): [[Image:BME103_Group13_SIForwardprimer.png]]
	<br>Reverse Primer (5' to 3'): [[Image:BME103_Group13_SIReverseprimer.png]]		<br>Reverse Primer (5' to 3'): [[Image:BME103_Group13_SIReverseprimer.png]]
		+	<br><br> A disease allele will produce a PCR product while a non-disease will not produce a PCR product due to the ability of the forward primer, which contains the mutated T, to bind to the reverse strand. If the reverse strand has a corresponding A at the position opposite the mutated T then the primer will bind. If the reverse strand has a C instead (normal), the forward primer will not bind to the reverse strand. This is illustrated in the figure below.
	<!--- Include the sequences of your forward and reverse primers. Explain why a disease allele will give a PCR product and the non-disease allele will not. --->		<!--- Include the sequences of your forward and reverse primers. Explain why a disease allele will give a PCR product and the non-disease allele will not. --->

Sudarshan Iyer: /* Research and Development */

Sudarshan Iyer — Thu, 29 Nov 2012 07:02:13 GMT

Research and Development

←Older revision		Revision as of 07:02, 29 November 2012
Line 176:		Line 176:
	'''Primer Design'''		'''Primer Design'''
	<br><br>Since this SNP lies on the forward strand, the primer used to facilitate a DNA signal of the mutated DNA will include a forward primer containing the mutation and a reverse primer 200 base pairs to the right of the mutation, with each primer being 20 base pairs long.		<br><br>Since this SNP lies on the forward strand, the primer used to facilitate a DNA signal of the mutated DNA will include a forward primer containing the mutation and a reverse primer 200 base pairs to the right of the mutation, with each primer being 20 base pairs long.
-	<br>Forward Primer (5' to 3'):	+	<br>Forward Primer (5' to 3'): [[Image:BME103_Group13_SIForwardprimer.png]]
-	<br>Reverse Primer (5' to 3'):	+	<br>Reverse Primer (5' to 3'): [[Image:BME103_Group13_SIReverseprimer.png]]
	<!--- Include the sequences of your forward and reverse primers. Explain why a disease allele will give a PCR product and the non-disease allele will not. --->		<!--- Include the sequences of your forward and reverse primers. Explain why a disease allele will give a PCR product and the non-disease allele will not. --->

←Older revision		Revision as of 18:55, 29 November 2012
Line 15:		Line 15:
	\| [[Image:Voltage_main.jpg\|100px\|thumb\|Name: Garrett Repp<br>Open PCR machine engineer]]		\| [[Image:Voltage_main.jpg\|100px\|thumb\|Name: Garrett Repp<br>Open PCR machine engineer]]
	\| [[Image:Joseph_pic.jpeg\|100px\|thumb\|Name: Joseph Rosario<br>Open PCR machine engineer]]		\| [[Image:Joseph_pic.jpeg\|100px\|thumb\|Name: Joseph Rosario<br>Open PCR machine engineer]]
-	\| [[Image:~~BME103student~~.jpg\|100px\|thumb\|Name: Ujwala Vaka<br>Experimental protocol planner]]	+	\| [[Image:dnaproblems.jpg\|100px\|100px\|thumb\|Name: Ujwala Vaka<br>Experimental protocol planner]]
	\| [[Image:BME103_Group13_emily.jpg\|100px\|thumb\|Name: Emily Herring<br>Experimental protocol planner]]		\| [[Image:BME103_Group13_emily.jpg\|100px\|thumb\|Name: Emily Herring<br>Experimental protocol planner]]
	\| [[Image:BME103_Group13_SIavatar.jpg\|100px\|thumb\|Name: Sudarshan Iyer<br>Research and Development scientist]]		\| [[Image:BME103_Group13_SIavatar.jpg\|100px\|thumb\|Name: Sudarshan Iyer<br>Research and Development scientist]]