BME103:T130 Group 10 - Revision history

Frances Lakers: /* Results */

Frances Lakers — Thu, 15 Nov 2012 21:46:47 GMT

Results

←Older revision		Revision as of 21:46, 15 November 2012
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	\| PCR: Water \|\| 7193042 \|\| 0 \|\| Negative (No Signal)		\| PCR: Water \|\| 7193042 \|\| 0 \|\| Negative (No Signal)
	\|-		\|-
-	\| PCR: Patient 1 ID ~~#####~~, rep 1 \|\| 6917330 \|\| 1.476 \|\| Negative	+	\| PCR: Patient 1 ID 92986, rep 1 \|\| 6917330 \|\| 1.476 \|\| Negative
	\|-		\|-
-	\| PCR: Patient 1 ID ~~#####~~, rep 2 \|\| 18548291 \|\| 3.958 \|\| Positive	+	\| PCR: Patient 1 ID 92986, rep 2 \|\| 18548291 \|\| 3.958 \|\| Positive
	\|-		\|-
-	\| PCR: Patient 1 ID ~~#####~~, rep 3 \|\| 9971894 \|\| 2.128 \|\| Positive	+	\| PCR: Patient 1 ID 92986, rep 3 \|\| 9971894 \|\| 2.128 \|\| Positive
	\|-		\|-
-	\| PCR: Patient 2 ID ~~#####~~, rep 1 \|\| 766751 \|\| 0.164 \|\| Negative (No Signal)	+	\| PCR: Patient 2 ID 81682, rep 1 \|\| 766751 \|\| 0.164 \|\| Negative (No Signal)
	\|-		\|-
-	\| PCR: Patient 2 ID ~~#####~~, rep 2 \|\| 6144131 \|\| 1.311 \|\| Negative	+	\| PCR: Patient 2 ID 81682, rep 2 \|\| 6144131 \|\| 1.311 \|\| Negative
	\|-		\|-
-	\| PCR: Patient 2 ID ~~#####~~, rep 3 \|\| 7734546 \|\| 1.650 \|\| Positive	+	\| PCR: Patient 2 ID 81682, rep 3 \|\| 7734546 \|\| 1.650 \|\| Positive
	\|}		\|}

Frances Lakers: /* Protocols */

Frances Lakers — Thu, 15 Nov 2012 21:13:37 GMT

Protocols

←Older revision		Revision as of 21:13, 15 November 2012
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	1.)'''HOW PCR WORKS'''		1.)'''HOW PCR WORKS'''

-	The Polymerase Chain Reaction machine, PCRm for short, works by cycling DNA through different temperatures to amplify the desired strand so the sample can be compared with other DNA. First, the DNA is denatured by heating the samples to such temperature that the hydrogen bonds holding the double-stranded molecule together are broken. The sample is then cooled, which allows a primer to bind to the target DNA. In the third step, the sample is heated back up so an enzyme can replicate the DNA. In the final step, a fluorescent dye binds to the new double-stranded DNA. This process is repeated many times, amplifying the target strand to an analyzable quantity.	+	The Polymerase Chain Reaction machine, PCRm for short, works by cycling DNA through different temperatures to amplify the desired strand so the sample can be compared with other DNA. First, the DNA is denatured by heating the samples to such temperature that the hydrogen bonds holding the double-stranded molecule together are broken. The sample is then cooled, which allows a primer to bind to the target DNA. In the third step, the sample is heated back up so an enzyme, in our case Taq Polymerase, can replicate the DNA. In the final step, a fluorescent dye binds to the new double-stranded DNA. This process is repeated many times, amplifying the target strand to an analyzable quantity.

	2.)'''STEPS TO AMPLIFY DNA'''		2.)'''STEPS TO AMPLIFY DNA'''

Jeffery C. Ramirez: /* Initial Machine Testing */

Jeffery C. Ramirez — Thu, 15 Nov 2012 19:36:27 GMT

Initial Machine Testing

←Older revision		Revision as of 19:36, 15 November 2012
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	==Initial Machine Testing==		==Initial Machine Testing==
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		+	'''PCR MACHINE'''
		+
		+	[[Image:OpenPCR1.jpg]]
		+
		+

Jeffery C. Ramirez: /* Protocols */

Jeffery C. Ramirez — Thu, 15 Nov 2012 15:52:37 GMT

Protocols

←Older revision		Revision as of 15:52, 15 November 2012
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	'''''Fluorimeter Measurements'''''<br>		'''''Fluorimeter Measurements'''''<br>
-


	1.)'''PHOTO OF SETUP'''		1.)'''PHOTO OF SETUP'''
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		+	[[Image:Fluorimeter.jpg\|500px\|thumb\|]]
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	2.)'''STEPS TO ASSEMBLE THE FLUORIMETER'''		2.)'''STEPS TO ASSEMBLE THE FLUORIMETER'''

Jeffery C. Ramirez: /* Protocols */

Jeffery C. Ramirez — Thu, 15 Nov 2012 15:46:47 GMT

Protocols

←Older revision		Revision as of 15:46, 15 November 2012
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	5.)'''SAMPLE DESCRIPTIONS'''		5.)'''SAMPLE DESCRIPTIONS'''

-	In this lab there were 8 samples that were tested. These samples include a positive control which has the cancer gene, a negative control which has no cancer gene and 3 tubes of DNA from two different subjects.	+	In this lab there were 8 samples that were tested. These samples include a positive control, which has the cancer gene; a negative control, which has no cancer gene and 3 tubes of DNA from two different subjects.
	<br>		<br>

Jeffery C. Ramirez: /* Protocols */

Jeffery C. Ramirez — Thu, 15 Nov 2012 09:24:48 GMT

Protocols

←Older revision		Revision as of 09:24, 15 November 2012
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	5.)'''SAMPLE DESCRIPTIONS'''		5.)'''SAMPLE DESCRIPTIONS'''
		+
		+	In this lab there were 8 samples that were tested. These samples include a positive control which has the cancer gene, a negative control which has no cancer gene and 3 tubes of DNA from two different subjects.
	<br>		<br>

Jeffery C. Ramirez: /* Protocols */

Jeffery C. Ramirez — Thu, 15 Nov 2012 09:19:07 GMT

Protocols

←Older revision		Revision as of 09:19, 15 November 2012
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	Step 3. After the series of cycles is completed, the PCRm temperature is then held at 72°C for 3 minutes.		Step 3. After the series of cycles is completed, the PCRm temperature is then held at 72°C for 3 minutes.

-	Step 4. The PCRm is then kept ~~constant~~ at 4°C	+	Step 4. The PCRm is then kept constnt at 4°C

	3.)'''COMPONENTS OF PCR MIX'''		3.)'''COMPONENTS OF PCR MIX'''
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	4.)'''TABLE OF REAGENTS AND VOLUMES USED'''		4.)'''TABLE OF REAGENTS AND VOLUMES USED'''
		+
		+	{\| {{table}}
		+	\| align="center" style="background:#f0f0f0;"\|'''Reagent'''
		+	\| align="center" style="background:#f0f0f0;"\|'''Volume'''
		+	\|-
		+	\| Template DNA (20 ng)\|\|0.2μL
		+	\|-
		+	\| 10 μM forward primer\|\|1.0μL
		+	\|-
		+	\| 10 μM reverse primer\|\|1.0μL
		+	\|-
		+	\| GoTaq master mix\|\|50.0μL
		+	\|-
		+	\| dH<sub>2</sub>O\|\|47.8μL
		+	\|-
		+	\| Total\|\|100μL
		+	\|}

	5.)'''SAMPLE DESCRIPTIONS'''		5.)'''SAMPLE DESCRIPTIONS'''

Jeffery C. Ramirez: /* Protocols */

Jeffery C. Ramirez — Thu, 15 Nov 2012 08:51:50 GMT

Protocols

←Older revision		Revision as of 08:51, 15 November 2012
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	Step 1. The PCRm lid is heated to 100°C and the sample tubes are heated to 95°C. This temperature is held constant for 3 minutes.		Step 1. The PCRm lid is heated to 100°C and the sample tubes are heated to 95°C. This temperature is held constant for 3 minutes.

-	Step 2. The PCRm is then set to run 30 ~~consecutive temperature~~ cycles. Each cycle consists of heating the sample to 95°C for 30 seconds, cooling to 57°C for 30 seconds, and finally, heating to 72°C for 30 seconds.	+	Step 2. The PCRm is then set to run 30 consecutivetemperature cycles. Each cycle consists of heating the sample to 95°C for 30 seconds, cooling to 57°C for 30 seconds, and finally, heating to 72°C for 30 seconds.

	Step 3. After the series of cycles is completed, the PCRm temperature is then held at 72°C for 3 minutes.		Step 3. After the series of cycles is completed, the PCRm temperature is then held at 72°C for 3 minutes.
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	3.)'''COMPONENTS OF PCR MIX'''		3.)'''COMPONENTS OF PCR MIX'''
		+
		+	The PCR mix consisted of the template DNA, a forward and reverse primer, the GoTaq master mix which is the enzyme, and finally some dH<sub>2</sub>O

	4.)'''TABLE OF REAGENTS AND VOLUMES USED'''		4.)'''TABLE OF REAGENTS AND VOLUMES USED'''

Frances Lakers: /* OUR TEAM */

Frances Lakers — Thu, 15 Nov 2012 08:01:12 GMT

OUR TEAM

←Older revision		Revision as of 08:01, 15 November 2012
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	\| [[Image:BME103_Group10_tylert.jpg\|100px\|thumb\|Name:<br> '''Tyler Tamasauckas''' <br>Role:<br> '''R&D Specialist''']]		\| [[Image:BME103_Group10_tylert.jpg\|100px\|thumb\|Name:<br> '''Tyler Tamasauckas''' <br>Role:<br> '''R&D Specialist''']]
	\| [[Image:BME103_Group10_AlexWhyYouNoUploadProfilePic.jpg‎\|100px\|thumb\|Name:<br> '''Alexander Baldwin''' <br>Role:<br> '''Open PCR Machine Engineer''']]		\| [[Image:BME103_Group10_AlexWhyYouNoUploadProfilePic.jpg‎\|100px\|thumb\|Name:<br> '''Alexander Baldwin''' <br>Role:<br> '''Open PCR Machine Engineer''']]
-	\| [[Image:BME103_Group10_FrancesL.jpg\|100px\|thumb\|Name:<br> '''Frances Lakers'''<br>Role:<br> '''~~R&D Specialist;~~ Data Analyzer''']]	+	\| [[Image:BME103_Group10_FrancesL.jpg\|100px\|thumb\|Name:<br> '''Frances Lakers'''<br>Role:<br> '''Data Analyzer''']]
	\|}		\|}

Frances Lakers: /* Results */

Frances Lakers — Thu, 15 Nov 2012 08:00:08 GMT

Results

←Older revision		Revision as of 08:00, 15 November 2012
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	\| PCR: Calf Thymus \|\| 9372391 \|\| 2 \|\| Positive		\| PCR: Calf Thymus \|\| 9372391 \|\| 2 \|\| Positive
	\|-		\|-
-	\| PCR: Water \|\| 7193042 \|\| 0 \|\| Negative	+	\| PCR: Water \|\| 7193042 \|\| 0 \|\| Negative (No Signal)
	\|-		\|-
	\| PCR: Patient 1 ID #####, rep 1 \|\| 6917330 \|\| 1.476 \|\| Negative		\| PCR: Patient 1 ID #####, rep 1 \|\| 6917330 \|\| 1.476 \|\| Negative
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	\| PCR: Patient 1 ID #####, rep 3 \|\| 9971894 \|\| 2.128 \|\| Positive		\| PCR: Patient 1 ID #####, rep 3 \|\| 9971894 \|\| 2.128 \|\| Positive
	\|-		\|-
-	\| PCR: Patient 2 ID #####, rep 1 \|\| 766751 \|\| 0.164 \|\| Negative	+	\| PCR: Patient 2 ID #####, rep 1 \|\| 766751 \|\| 0.164 \|\| Negative (No Signal)
	\|-		\|-
	\| PCR: Patient 2 ID #####, rep 2 \|\| 6144131 \|\| 1.311 \|\| Negative		\| PCR: Patient 2 ID #####, rep 2 \|\| 6144131 \|\| 1.311 \|\| Negative
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	KEY		KEY
-	* '''Sample''' = <!--- explain what "sample" means --->	+	* '''Sample''' = The specific genetic material used in that trial. For example, the three trials labeled Patient 1 used DNA from that patient and that patient only.<!--- explain what "sample" means --->
-	* '''Integrated Density''' = <!--- explain what "integrated density" means and how you did background subtraction to get this value --->	+	* '''Integrated Density''' = The sum of the values of the pixels in the sample image area. Also equivalent to product of the Mean Gray Value and the Area. The above values represent the integrated density after removing the "background noise" of the image's background pixel values. In math terms, Net IntDen = (IntDen Sample) - (IntDen Background)<!--- explain what "integrated density" means and how you did background subtraction to get this value --->
-	* '''DNA μg/mL''' = <!--- explain how you calculated this --->	+	* '''DNA μg/mL''' = Amount of genetic material present within sample. This was calculated using a proportional equation. Given that the Calf Thymus sample contained 2μg/mL, we were able to set up this equation for estimating the amount of genetic material present in the unknown samples. <br>(Sample)μg/mL = (IntDen Sample)*[(2μg/mL)/(IntDen Calf Thymus)]<!--- explain how you calculated this --->
-	* '''Conclusion''' = <!--- explain what "Positive" and "No signal" means, relative to the control samples --->	+	* '''Conclusion''' = Our conclusion as to whether or not the sample genetic material contains the polymorphism for cancer. A "Positive" conclusion indicates a likelihood of the cancerous polymorphism being present while a "Negative" or "No Signal" conclusion indicates a likelihood of the polymorphism's absence. These conclusions were mainly drawn based on the visual fluorescence of the samples and supported by the amount of genetic material present.<!--- explain what "Positive" and "No signal" means, relative to the control samples --->