BME100 f2013:W1200 Group14 L4

BME 100 Fall 2013	Home People Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3 Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6 Course Logistics For Instructors Photos Wiki Editing Help
OUR TEAM Name: Vaasavi Sundar Research and Development Name: Brie Schilling Open PCR Machine Testing Name: Minh Pham Open PCR Machine Testing Name: Ashley Powell Protocol Planning Name: Hannah Brutsche Protocol Planning Name: student Role(s) LAB 1 WRITE-UP Initial Machine Testing The Original Design The PCR is a device used by scientists to copy DNA strands and amplify them. With the usage of heating and cooling, DNA strands are separated. With the addition of polymerases, certain segments are copied multiple times in order to observe and diagnose diseases, viruses, and bacteria. Experimenting With the Connections When we unplugged (part 3) from (part 6), the display screeen turns off and shows nothing at all. When we unplugged the white wire that connects (part 6) to (part 2), the temperature sensor malfunctions and displays the incorrect temperature on the LCD screen. Test Run (Write the date you first tested Open PCR and your experience(s) with the machine) Protocols DNA Sample Set-up Positive Control: Cancer DNA Template, Tube Label: CT Patient 1 ID: 30337 Replicate 1 Tube Label: 303 Patient 1 ID: 30337 Replicate 2 Tube Label: 304 Patient 1 ID: 30337 Replicate 3 Tube Label: 305 Negative Control: Non-Cancer DNA Template, Tube Label: NCT Patient 2 ID: 57707 Replicate 1 Tube Label: 577 Patient 2 ID: 57707 Replicate 2 Tube Label: 578 Patient 2 ID: 57707 Replicate 3 Tube Label: 579 Materials PCR Reaction mix, 8 tubes (50 micrometers), DNA primer mix, disposable pipette DNA Sample Set-up Procedure 1) Label all different tubes so we don't mix up any of the results. 2) Add 25μL of DNA sample mix to each corresponding tube, using different disposable pipette tips for each tube. 3) Place the tubes into a thermocycler 4) Program the Thermocycler to run as follows so PCR occurs. Thermocycler Program 1. 1 Cycle at 95°C for 3 minutes 2. 35 Cycles at 95°C for 30 seconds, 57°C for 30 seconds, 72°C for 30 seconds 3. 72°C for 3 minutes 4. Hold the mix at 4°C PCR Reaction Mix The PCR Reaction mix has Taq DNA Polymerase, MgCL2, and dNTP's. DNA/ primer mix The DNA primer mix has the same forward primer and reverse primer for all tubes Research and Development PCR - The Underlying Technology (Add a write-up, essay-style, organized into paragrpahs with descriptive headers, based on the Q&A's from Section three of your worksheet) PCR, or polymerase chain reaction, is used to amplify DNA to diagnose disease, map the human genome, or even to solve crimes. There are several components involved in completing PCR-- the template DNA, the primers, the Taq polymerase, magnesium chloride, and deoxyribonucleotides. The purpose of each component is as follows: Template DNA: The template DNA contains the target sequence, and is the basis for the amplification. Without the template, the target sequence cannot be amplified, and the primers have nothing to bind to, and polymerase has nothing to copy. Primers: The primers bind to each single-stranded DNA and functions almost as a signal for Taq polymerase to begin the construction of the complementary sequence. Furthermore, the presence of both forward and reverse primers ensures that the target sequence, in particular, will be amplified, because the primers are made specifically for the sections of the template DNA which include the target sequence. Taq polymerase: This is an enzyme which constructs the complimentary sequence to the single strands of the template DNA. Magnesium chloride: The magnesium chloride acts as a catylyst for the polymerase reaction, and helps the reaction move along faster. Deoxyribonucleotides: These are free-floating nucleotides, and these are the bases that are used by Taq polymerase to create the complimentary sequence for the single-stranded DNA. The aforementioned materials are the key components in a PCR reaction. The first step of PCR is to heat the DNA to around 95 degrees celsius for around 3 minutes, and this allows the DNA to denature and unwind into two separate strands. The temperature, which is near boiling, and the required time, are both imperative to this reaction because this is the amount of energy that is required to break the hydrogen bonds between the nucleotides of the complementary strands. Once the DNA denatures, the temperature must be lowered to 57 degrees for around 30 seconds. The lowering of temperature allows for the forward and reverse primers to bind onto each single stranded-DNA. After 30 seconds of this, the temperature is once again re-heated to 72 degrees, the ideal temperature for Taq polymerase. Taq polymerase now begins constructing complementary strands to each single template strand. This continues for around 3 minutes. This cycle repeats itself over and over, until the "target sequence" is amplified more so than the original template DNA. The strands are complementary, and are created with paired nucleotides-- adenine binds with thymine, and cytosine with guanine. Base pairing, which is done through hydrogen bonds, are what create the "spiral staircase" of DNA structure. This is also what allows primers to the template strand. (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 PCR video/ tutorial might be useful. Be sure to credit the sources if you borrow images.)