BME103:T930 Group 1

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Results)
(Results)
Line 115: Line 115:
<!--- Place two small Image J data images here. One showing the result of Water and the other showing the result of Calf Thymus DNA --->
<!--- Place two small Image J data images here. One showing the result of Water and the other showing the result of Calf Thymus DNA --->
-
 
+
[[Image:BME103_Group1_water.jpg‎|250px|]]<br>
<!--- Enter the values from your group's Data Analyzer table below. E6, F6, etc. are the excel cells from which you should copy your data. --->
<!--- Enter the values from your group's Data Analyzer table below. E6, F6, etc. are the excel cells from which you should copy your data. --->

Revision as of 14:43, 14 November 2012

BME 103 Fall 2012 Home
People
Lab Write-Up 1
Lab Write-Up 2
Lab Write-Up 3
Course Logistics For Instructors
Photos
Wiki Editing Help
Image:BME494_Asu_logo.png

Contents

OUR TEAM

Joseph Heath:Research & Development Scientist & PCR Machine Engineer
Joseph Heath:
Research & Development Scientist & PCR Machine Engineer
Jessica Kemper:Experimental Protocol Planner
Jessica Kemper:
Experimental Protocol Planner
Maile Ravenkamp:Experimental Protocol Planner
Maile Ravenkamp:
Experimental Protocol Planner
Nick Hool:PCR Machine Engineer
Nick Hool:
PCR Machine Engineer
Christian Boden:PCR Machine Engineer & Research & Development Scientist
Christian Boden:
PCR Machine Engineer & Research & Development Scientist

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design
Image:Something that makes sense.jpg


This machine is called an Open PCR machine. PCR stands for polymerase chain reaction, and this machine helps us create specific strands of DNA. It can hold 16 tubes of DNA and is compatible with any computer that has the appropriate downloaded software. The machine goes through a series of steps to recreate the DNA. First, it heats up to break apart the DNA strands. Then, it cools down to allow polymer chains to attach to the target DNA sequence. Once the primers are attached, the machine will heat up again so that the protein in charge of DNA construction will activate and bind to the polymers and then start to build the DNA sequences that are targeted. This machine is capable of replicating millions of segments of a specific DNA sequence in just an hour or two. This machine, like many others, can be improved but for standard use, this machine works fine as is. If we improve this machine, the process that the machine goes through will most likely be the same but the hardware of the Open PCR machine may be changed. For example, if we wanted to make the amount of time for each cycle shorter, we could improve the heating elements of the machine so that the heating and cooling will be faster and more effective.


Experimenting With the Connections

When we unplugged the mounting plate from the open PCR circuit board, the display screen on the PCR box did not work.

When we unplugged the white wire that connects the open PCR circuit board to the heating block, there was no temperature reading on the display screen.


Test Run

(First Open PCR test: 10/25/12. We had a successful and simple run of PCR)




Protocols

Polymerase Chain Reaction

Components of the PCR Master Mix

1. Modified Taq DNA polymerase
2. dNTP's
3. MgCl2
4. reaction buffers

1. Thaw the GoTaq Colorless Master Mix at room temperature. Vortex the Master Mix, then spin it briefly in a microcentrifuge to collect the material at the bottom of the tube.

2. Prepare the following reaction mix on ice:

Reagents and Volumes used in PCR replication
Reagents and volumes used in PCR replication

3. If using a cycler without a heated lid, overlay the reaction mix with 1-2 drops of mineral oil to prevent evaporation during thermal cycling. Centrifuge the reaction mix in a microcentrifuge for 5 seconds.

4. Place the reactions in a thermal cycler that has been preheated to 95 degrees Celsius. Perform PCR.

How To Amplify A Patient's DNA Sample
1. Denaturation: a 2-minute denaturation at 95 degrees celsius.

2. Annealing: perform the reaction about 5 degrees Celsius below the calculated melting temperature of the primers and increasing the temperature in increments of 1°C to the annealing temperature; this should occur anywhere between 30 seconds and 1 minute.

3. Extension: performed between 72-74 degrees Celsius, extension allows 1 minute for every 1 kb of DNA to be amplified; the suggested time for extension is 5 minutes.

4. Refrigeration: refrigerate the tubes at 4 degrees Celsius for several hours; this will minimize the opportunity for DNA polymerase to continue to be active at higher temperatures.

5. Cycle Number: the optimal amplification is 25-30 cycles, but up to 40 may be performed.

Flourimeter Measurements

Flourimeter Procedure

1. turn on the excitation light using the switch for the Blue LED.
2. Place your smart phone on the cradle at a right angle from the slide.
3. Turn on the camera setting on the smartphone. Turn off the flash and set the ISO to 800 or higher and increase the exposure to maximum. You should also turn off the autofocus, if possible, and make sure that you can take an image where the drop on the slide will be in focus.
4. Adjust the distance between the smartphone on its cradle and the first two rows of the slide so that it is as close as you can get without having a blurry image.
5. The pipette should be filled with liquid only to the bottom of the black line. Then, carefully place two drops of water in the middle of the first two rows of the slide using the plastic pipette. Then add two more drops. The drop should then be pinned and look like a beach ball. It should be between 130-160 μL.
6. Align the drop by moving the slide so that the blue LED light is focused by the drop to the middle of the black fiber optic fitting on the other side of the drop (you will see that it has a small opening that is used for spectral measurement).
7. Cover the fluorimeter with the light box, but make sure you can access your smartphone to take the image. The light box should be used to remove as much stray light as possible, but do not worry if you have some light.
8. Take three images of the drop of water. Do not move your smartphone.
9. Remove the box and be careful not to move your smartphone. If you want to adjust for any movement, use the ruler provided to measure the distance so that you can return to that location. You can also use ImageJ to compensate for moving the camera, but it makes the analysis more complicated.
10. Use a clean plastic pipette to remove the water drop from the surface.
11. Push the slide in so that you are now in the next set of two holes.
12. Repeat steps 5-10 four more times so that you have now imaged all 5 positions on the slide.
13. Record the type of smartphone you used, the distance from the base of smartphone cradle to measurement device, and attach one image for each position of the drop.
Patients and Samples
Samples
Patients and Samples
Sample Labels and Patient Numbers



Research and Development

Specific Cancer Marker Detection - The Underlying Technology

The r17879961 cancer-associated sequence (AAACTCTTACACTGCATACA) will produce a DNA signal because of its nucleotide variation (ACATTGC to ACACTGC). This T-C change results in an isoleucene to threonine substitution. In a study in Finland, patients with colorectal cancer (CRC), the most common cancer associated with the DNA sequence change, had the allele 7.8% of the time while patients without CRC had the allele in 5.3% of patients, showing a significantly higher association in CRC patients.[1] PCR detection will only give a signal if this allele is present.



Results


Sample Integrated Density DNA μg/mL Conclusion
PCR: Negative Control 13908255 F6 clear
PCR: Positive Control 27282151 F7 green
PCR: Patient 1 ID 43417, rep 1 35526894 F8 green
PCR: Patient 1 ID 43417, rep 2 19073943 F9 green
PCR: Patient 1 ID 43417, rep 3 29391013 F10 green
PCR: Patient 2 ID 11260 , rep 1 1903450 F11 clear
PCR: Patient 2 ID 11260, rep 2 5214727 F12 slightly green
PCR: Patient 2 ID 11260, rep 3 5099077 F13 slightly green


KEY

  • Sample = Describes sample number and patient
  • Integrated Density = The Integrated Density of the Drop minus that of the background
  • DNA μg/mL = 2 * IntDen of Sample/IntDen of Calf Thymus
  • Conclusion = Whether exponential DNA replication has occured
Personal tools