BME100 s2015:Group8 12pmL4

BME 100 Spring 2015	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: Priscilla Hernandez Name: Alwaleed Alsahafi Name: Victoria Sanford Name: Sarah Soaf Name: Taylor Simone Woods LAB 4 WRITE-UP Protocol Materials Lab coat and disposable gloves. PCR reaction mix, 8 tubes, 50 μL each: Mix contains Taq DNA polymerase, MgCl2, and dNTP’s (http://www.promega.com/resources/protocols/product-information-sheets/g/gotaq- colorless-master-mix-m714-protocol/) DNA/ primer mix, 8 tubes, 50 μL each: Each mix contains a different template DNA. All tubes have the same forward primer and reverse primer. A strip of empty PCR tubes. Disposable pipette tips: only use each only once. Never re-use disposable pipette tips or samples will be cross-contaminated. Cup for discarded tips. Micropipettor. OpenPCR machine: shared by two groups PCR Reaction Sample List Tube Label PCR Reaction Sample Patient ID G# + Positive control G# - Negative control G# 1-1 Patient 1, replicate 1 62525 G# 1-2 Patient 1, replicate 2 62525 G# 1-3 Patient 1, replicate 3 62525 G# 2-1 Patient 2, replicate 1 86787 G# 2-2 Patient 2, replicate 2 86787 G# 2-3 Patient 2, replicate 3 86787 DNA Sample Set-up Procedure Step 1 Retrieve two patient samples (with given patient ID) and negative and positive control from the TA. The negative control is be a patient who has tested negative to having the SNP gene and the positive control is a patient who has tested positive. There will be three tubes for each patient. Step 2 Make labels for the tubes with your group number and the corresponding label patient replicate. For example, this group's would label patient one's first replicate G82 1-1. Step 3 Write down the patient ID's in their proper location in the table, but not on the tubes. OpenPCR program HEATED LID: 100°C INITIAL STEP: 95°C for 2 minutes NUMBER OF CYCLES: 35 Denature at 95°C for 30 seconds, Anneal at 57°C for 30 seconds, and Extend at 72°C for 30 seconds FINAL STEP: 72°C for 2 minutes FINAL HOLD: 4°C Research and Development PCR - The Underlying Technology PCR consists of five steps and a holding phase, three of the steps are cycled 35 times. A PCR solution will require four main ingredients. The first ingredient that it will require is a template DNA source. The source nor the organism that it came from matters but purer DNA yields higher results. Primers which are short single-strand segments of DNA are necessary to bind to the complementary segment of the single-stranded DNA and for Taq polymerase to extend upon, thus replicating and amplifying the sought after DNA segment. The final component deoxyribonucleotides or dNTP's are needed for the polymerase to base pair the template strand with.Taq Polymerase which has been derived from a thermophile is used instead of regular DNA polymerase because it works optimally at 72 degrees Celsius which is the optimal temperature for the extension step. Most PCR solutions will also contain a reaction buffer in order to maintain optimal pH which will also maximize yields. During the first initial step, the PCR mix will be held at 95 degrees Celsius for 3 minutes. The high heat breaks the hydrogen bond that exist between the two stands of DNA leaving them as two single strands of DNA. This step is only repeated once. The next three steps are each 30 seconds and are cycled 35 times, starting with the denature step at 95 degrees Celsius which seems repetitive the first time but will serve to separate the primers from the single strand DNA so that the the next two steps may once again occur. In the anneal step the mix is held at 57 degrees Celsius so that the primers may bond to their complementary DNA segment. Two primers are made so that first, oriented in the forward direction and the second, in the reverse direction will contain the segment meant for amplification in between. The final cycled step is the extension step which is at 72 degrees Celsius, in this step Taq polymerase binds to the two primers and starts extending them. This extension is done by Taq polymerase grabbing a free floating dNTP that complements the base immediately following the primer and bonding it with the template strand. This is repeated until it meets the Taq polymerase coming in the opposite direction. The final extension step which is once again 72 degrees Celsius but for three minutes lets any partially replicated strands finish. It is held at 4 degrees Celsius until retrieval time. Taq polymerase follows very strict restrictions for base pairing. In other words it doesn't match randomn nucleotides. Purines are always matched with a pyrimidine.