BME103:T130 Group 17 l2

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Contents

OUR TEAM

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LAB 2 WRITE-UP

Thermal Cycler Engineering

Our re-design is based upon the Open PCR system originally designed by Josh Perfetto and Tito Jankowski.


System Design
Image of the heating block

Image of the cooling system


Key Features
The first picture shows the heating block that is in use in the current PCR machine. It is limited in that it can only contain 16 test tubes at any given time. Our redesign calls for doubling the amount of test tube holders by increasing the size of the heating block. The picture below is that of the current cooling system. Because our redesign is going the be running PCR on twice as many samples, an increase in the cooling power needs to occur in order to sufficiently cool the larger number of samples. This is done by increasing the size of the cooling system and therefore increasing its power.


Instructions
1) Remove the current heating block and cooling system from the PCR machine.
2) Replace removed items with the larger heating block and larger cooling system.
3) Replace side panels with larger ones in case any of the replaced parts exceed the confines of the current PCR machine.
4) Continue to run PCR as normal.




Protocols

Material Tables

Supplied in the Kit Amount
Pre-labeled PCR tubes 32 tubes
Pre-labeled bulb pipets 34 pipets
20 µM forward primer (ALZ) 2.0 μL
20 µM reverse primer (ALZ) 2.0 μL
GoTaq Master Mix 100.0 μL
dH2O 95.6 μL
Total Volume 199.6 μL
Supplied by the user Amount
DNA (40 ng) 0.4 μL
Computer 1
Lab coats 1 per person
gloves 2 per person

PCR Protocol



DNA Measurement Protocol

1. Turn on the blue light in the Flourimeter using the switch for the Blue LED.

2. Place the smart phone in the cradle provided at the right angle and distance from the slide.

3. Turn on the camera on the phone. TURN OFF THE FLASH. Set the ISO to 800 or higher. Increase the exposure to maximum.

4. Adjust the distance between phone and the first two rows of dots of the slide so that the picture will be clear and the camera does not give a blurred image.

5.First label the blank pipettes according to the patients (A,B,C,D... all eight of them). The pipettes given by the instructor are color coded. The white coded pipette is used for water. the red coded pipette is used for the calibrator (the tube with the red dot). The blue coded pipette is used for the sybrgreen (the tube with the blue dot). The black coded pipette is used to pick up the waste and put it in the cup that collects the waste droplets.

6. Align the slide so that the blue dot passed between the first two dots on the slide in the middle column. First calibrate the machine (to make sure the machine works). To calibrate the machine first put two drops of water on each dot and if the droplets are not connected then add a third droplet to combine the two separate droplets to one droplet. Put two droplets of the cyber green on the first two dots in the middle.

7. Then set the smart phone accordingly to take a clear picture of the droplet. Then put the black box on top of the phone and the machine so that when the the light is completely blocked and the shade of blue of green is shown when the picture is taken. 8. Record observations. 9. Remove the solution from the glass dish using the black pipette and discard it in the plastic cup given for the waste droplets. 10.Repeat step 6-9, but instead of adding calibrator solution, add 2 droplets of water. 11.Repeat steps 6-9 for patient 1 solutions (A,B,C,D) and patient 2 solutions (A2.B2,C2,D2).

Research and Development

Background on Disease Markers



Primer Design



Illustration


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