BME103:T130 Group 3 l2

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
OUR TEAM Name: Serena Kaplan Research and Development Name: Gabe McInnis Open PCR Machine Engineer Name: Blake Eichler Experimental Protocol Planner Name: Sierra Morris Experimental Protocol Planner Name: Zazu Moloi Open PCR Machine Engineer Name: Katelin Vaughn Research and Development 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 Key Features The specific feature being focused on to improve the Open PCR Machine is the heating and cooling aspect of the PCR machine. The main alteration of the machine would be to change the material used on the heat sink and heating pad to copper. This would allow for a much quicker change in temperature while the machine is running. Therefore, the heating and cooling phases of the cycles would be attained faster, thus cutting the time of a full PCR efficiently. The material change from a zinc alloy to copper would make heating and cooling the cycles of the PCR machine much quicker because copper is the most electrically conductive metal. Instructions There was only a change in the material of the heat sink and heating pad so the assembly instruction will remain the same as the original design. Protocols Materials Supplied in the Kit PCR Machine 10μM forward primer 10μM reverse primer GoTaq master mix Supplied by the User Template DNA Distilled Water Pipets Eppendorf Tubes PCR Protocol 1.) Pipet 0.1μL of template DNA, 0.5μL of 10μM forward primer, 0.5μL of 10μM reverse primer, 25.0μL of GoTaq Master Mix, and 23.9μL of dH20 in an Eppendorf tube. All of these items mixed together should create a total volume of 50.0μL. 2.) Up to 16 of these samples are then loaded into the PCR machine 3.) The DNA samples are then heated to ninety-five degrees Celsius (95°C) for one (1) minute in order to unzip the two single strands. 4.) They are then cooled to fifty-seven degrees Celsius (57°C) for ten (10) seconds so that the primers can attach to their matching sequences. 5.) Finally, they are heated back to seventy-two degrees Celsius (72°C) for ten (10) seconds, and polymerase extends the DNA strands by attaching the correct free nucleotides in order on the single strands. DNA Measurement Protocol Fluorimeter Setup 1.) The lid was first taken off the box and one of its sides was unbuttoned in order to create a flap. 2.) The box was then flipped upside down in order to create a dark environment for the camera. 3.) A hydrophobic slide was then inserted into the fluorimeter. 4.) Finally, the camera phone was placed in the stand. Fluorimeter Measurements 1.) Label transfer pipettes and tubes 2.) Transfer each sample separately into a tube containing 400μl of buffer 3.) Take the specifically labeled tube containing SYBR GREEN 1 and place two drops on the first two centered drops 4.) Place two drops of diluted sample on top of the SYBR GREEN 1 drop 5.) Align light through drop 6.) Take pictures using light box 7.) Repeat for each sample. 8.) Run water as BLANK using same procedure ImageJ Instructions 1.) Open ImageJ 2.) Click ANALYZE tool bar and select SET MEASUREMENTS 3.) Select AREA, MEAN GREY VALUE, and INTEGRATED DENSITY 4.) Upload image to ImageJ 5.) Select IMAGE then COLOR and then SPLIT CHANNELS 6.) Only use green channel 7.) Use OVAL tool and select the entire drop of liquid 8.) Go to ANALYZE and then MEASURE 9.) Drag circle to the background of the image 10.) Record results 11.) Repeat if necessary Research and Development Background on Disease Markers The single nucleotide polymorphism (SNP), 137852571, that is being examined in this experiment is linked with Androgen Insensitivity Syndrome and Kennedy Spinal and Bulbar Muscular Atrophy. Androgen Insensitivity Syndrome occurs when a person who is genetically male (who has one X and one Y chromosome) is resistant to male hormones (called androgens). As a result, the person has some or all of the physical traits of a female, but the genetic makeup of a male. The mutation on the X chromosome makes the body unable to respond to the hormones that produce a male appearance. Kennedy Spinal and Bulbar Muscular Atrophy is a debilitating neurodegenerative disease resulting in muscle cramps and progressive weakness due to degeneration of motor neurons in the brain stem and spinal cord. The SNP is located on the X chromosome and affects the gene AR, the gene is inherited in an x-linked recessive manner therefore only males can be fully affected by the mutation and females are rarely affected. The sequence of this gene is: CTTCTCCAGGCTTCCGCAACTTACAC[A/G]TGGACGACCAGATGGCTGTCATTCA The error in this sequence is represented by [A/G] which means that the normal G base pair has been mutated into an A base pair resulting in an allele that expresses the linked diseases. Breast Cancer rs137852571 AR – Reverse Primer: SNP:5,255,325 Missense GTG→ATG V[Val]→M[Met] Chromosome X Primer Design Normal: G mutates into cancer A CAACTTACACATGGACGACC reverse primer: GTTGAATGTGTACCTGCAGG Forward primer starts at 5,255,175: AGGGGTGGTGGGGAATTACC Illustration