BME103 s2013:T900 Group7

BME 103 Spring 2013	Home People Lab Write-Up 1 Lab Write-Up 2 Lab Write-Up 3 Course Logistics For Instructors Photos Wiki Editing Help
Name: Amanda Seaney Role(s) Name: Carissa Henriksen Role(s)Protocol Name: Nathan Scheuer Role: Open PCR machine Engineer Name: Samantha Barker Role:Research and Development Scientist Name: student Role(s) Name: student Role(s) LAB 1 WRITE-UP Initial Machine Testing [[|thumb|right|1600x1250x|]] The Original Design The picture to the right shows the design of the open PCR machine used in class. This PCR machine is compact, inexpensive, and easy to use in comparison to professional grade PCR equipment making it ideal for student and school use. The machine is composed of an LCD screen, heating lid, mother board (circuit board),thermal conductor and a fan. Experimenting With the Connections When we unplugged (part 3) from (part 6), the machine ... (did what? fill in your answer) When we unplugged the white wire that connects (part 6) to (part 2), the machine ... (did what? fill in your answer) Test Run (Write the date you first tested Open PCR and your experience(s) with the machine) Protocols Thermal Cycler Program For the reaction to take place, the machine needs to have a certain program set up to run. The program for the Thermal Cycler is as follows: 1 Cycle at 95 degrees Celcius for 3 minutes 35 Cycles at 95 degrees Celcius for 30 seconds, 57 degrees Celcius for 30 seconds, 72 degrees Celcius for 30 seconds 72 degrees Celcius for 3 minutes Hold the mix at 4 degrees Celcius Each cycle consists of denaturation, annealing, and extension. The denaturation step is when the DNA is heated to a point at which the double-stranded DNA comes apart into two single strands. Then the machine is cooled for the annealing step, which is when the primers attach to their respective sites on the single stranded DNA. The third step, extension, takes place at a warmer temperature than annealing, but cooler than denaturation, and consists of the elongation of the DNA strands. DNA Sample Set-up Tube: A, Positive Control: Cancer DNA template Tube: B, Patient 1 ID: 43825, Replicate: 1 Tube: C, Patient 1 ID: 43825, Replicate: 2 Tube: D, Patient 1 ID: 43825, Replicate: 3 Tube: E, Negative Control: No DNA template Tube: F, Patient 2 ID: 12079, Replicate: 1 Tube: G, Patient 2 ID: 12079, Replicate: 2 Tube: H, Patient 2 ID: 12079, Replicate: 3 DNA Sample Set-up Procedure To keep the patient samples separate and to avoid contamination between samples, it is necessary for one to label the tubes by numbering them one through eight with a permanent marker. One should take up 25 microliters of patient DNA into the micro pipet. Following this, one should add it to the 25 microliters of the PCR reaction mix. After all 8 tubes are filled with 50 microliters one should place them into the PCR machine, which is connected to the computer. PCR Reaction Mix One must create a PCR reaction mix, in order to do this groups are presented with 8 tubes, which will be filled with 25 microliters of the following: Taq DNA polymerase dNTPs Magnesium chloride (MgCl2) Premium concentrations for the maximum amount of DNA produced by PCR DNA/ primer mix One must create DNA/ primer mix, in order to do this groups are presented with 8 tubes, which will be filled with 25 microliters of the following: Unique DNA template of each patient Forward and reverse primers (the same contents in each tube) Research and Development Specific Cancer Marker Detection - The Underlying Technology (Add a write-up of the information discussed in Week 3's class) (BONUS points: Use a program like Powerpoint, Word, Illustrator, Microsoft Paint, etc. to illustrate how primers bind to the cancer DNA template, and how Taq polymerases amplify the DNA. Screen-captures from the OpenPCR tutorial might be useful. Be sure to credit the source if you borrow images.)