Name: Nicholas Sterkowitz Open PCR machine engineer
Name: Dominic Ilardi Open PCR machine engineer
Name: Alexandra Nazareno Experimental Protocol Planner
Name: Amanda Sweig Experimental Protocol Planner
Name: Taylor Deegan Research and Development(s)
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
To begin with we will change the frame from a wooden construction to recycled aluminum. This will maintain the light-weight portability of the machine, but will greatly reduce the fire hazard of heating a wooden box to the required temperatures. We will also add an additional row and column to the heating block to increase the product output without too much change to the overall size or cost of the machine.
We will also add two carrying handles to either side of the machine. Because the machine is somewhat awkward to carry and hold the handles will greatly reduce the chances of dropping if they are properly used.
Key Features
Instructions
Protocols
Materials
Supplied in PCR Kit
Amount
PCR Machine
1
Fluorometer Box
1
Calf Thymus
0.5 mL
SYBR Green (diluted)
1.5 mL
Camera Stand
1
Glass Slides
4
Calibration DNA
0.5 mL
GoTaq Polymerase and Buffer Solution
10 mL
Small Test Tubes
25
Large Test Tubes
25
Supplied by User
Amount
Smartphone Camera
1
Laptop
1
ImageJ Download
1
Power Supply
1
DNA Sample
At least 3 samples
PCR Protocol
1. Using a micro-pipette, transfer 1.0-1.5 mL of desired DNA sample into at least three test tubes.
2. reagent solution
3. place tubes into holder in PCR machine
4. program cycles on OpenPCR
5. Run reaction
DNA Measurement Protocol
1. Remove lid from black box and invert the box, open the front flap.
2. Place one glass slide into the holder.
3. Depending on the amount of samples being used, take a clean pipette for each sample and label it so that it is only used for that sample of DNA. For example, label a pipette for SYBR green with a horizontal line on the pipette bulb, and label a pipette for diluted water with a vertical line on the bulb. Do NOT use pipettes for any other solution than what they are labelled for.
4. Using the designated pipette, squeeze two drops of SYBR green dye inside the round glass windows on slide, preferably the second dot of the second row.
5. Begin by adding a sample of diluted water to the SYBR green dye, and using the appropriate labelled pipette, add two drops in the same spot as the SYBR green dye.
6. Align the sample so the blue LED shines directly though it focusing the light on the other side.
7. Move holder apparatus inside the box so that no light reaches it.
8. Place smartphone with the flash off onto holder and direct camera lens at the slide apparatus.
9. Take photo with smartphone and process on computer using ImageJ.
10. Using a clean pipette, remove the sample of diluted water and SYBR green dye from the slide.
11. Repeat steps 4-10 using samples of DNA, and moving back on the glass slide two rows each time a new sample is used.
12. Once there is no more rows on the glass slide, carefully discard the used slide and begin using a new one.
Research and Development
Background on Disease Markers
The SNP chosen for the lab is linked with Alzheimer's disease. Alzheimer's disease is the most common form of dementia. It has no cure and worsens as it progresses. Dementia is a loss of brain function and Alzheime's disease affects memory, thinking, and behavior.The associated SNP's for an increase in risk of Alzheimer's disease is rs1050283, which is located on the 12 chromosome.Information avaliable from the NCBI website:
http://www.ncbi.nlm.nih.gov/snp?term=rs1050283
Primer Design
The sequence that is connected with an increase risk of Alzheimer's disease is GGCTGGGCOCGGACATGGAGGACGTG[C/T]GCGGCCGCCTGGTGCAGTACCGCGG. The increase risk comes from the base change C to a T.
The reverse primer would be GGAGGACGTG[T]GCGGCCGCCT and the forward primer would be CCTCCTGCAG[A]CGCCGGCGGA.
A diseased allele will produce a PCR product because the gene associated with an increase risk that is trying to be tested for will have a T base in place of the normal C base. If the gene has this change in bases then the primers will be able to attach to the strands of DNA because the primers are designed to only attach to that specific sequence.
BME 103 Fall 2012
