BME100 s2015:Group2 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: Levi Riley Name: Alexandria Clark Name: Blossom Mendonca Name: Alina Kilic Name: Trevor Douglass Name: Andre Dang 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/gotaqcolorless-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 G2 + Positive control none G2 - Negative control none G2 1-1 Patient 1, replicate 1 58575 G2 1-2 Patient 1, replicate 2 58575 G2 1-3 Patient 1, replicate 3 58575 G2 2-1 Patient 2, replicate 1 86157 G2 2-2 Patient 2, replicate 2 86157 G2 2-3 Patient 2, replicate 3 86157 DNA Sample Set-up Procedure The group obtained all materials listed in the protocol section The group took the empty strip of PCR tubes given & cut them in half so that there were two strips of four linked tubes. This was done so that the PCR tubes would fit into the PCR machine because the machine only fits up to four in a row. The group labeled the empty PCR tubes using the labels from the table listed above. It was labeled using a black marker which was also erasable so that it could be removed afterwards. The tubes were transferred to a rack to secure them while the micro-pipette was used. In each of the eight tubes, 50 micro liters of PCR reaction mix was transferred into each tube. Then, in the tube labeled for positive control, 50 micro liters of solution was transferred. Similarly, 50 micro liters of solution was pipetted into the test tube labeled for the negative control. Between each use of the pipette, the tip was changed out to avoid contamination. After this was completed, 50 micro liters of the sample from patient 1 was transferred into it's appropriately labeled test tube. This same process was repeated for the remaining samples of patient one and patient two. The group took note to change the tip after each use of the pipette to avoid contamination. The group took note that all the labeled tubes to be transferred into the PCR machine contained 100 micro liters of material. The group closed all the lids for the PCR Reaction tubes (eight total). The group then asked the TA for help and placed the labeled PCR tubes into the appropriate rows for the PCR machine. A second group placed their PCR tubes into the same machine, and then the machine was set to the specifications listed below in the OpenPCR Machine section below. 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 Q1: what is the function of each Template DNA: The sample DNA that contains the target sequence, which is being amplified. The heat is added to separate the strands of the original double strand. Primers: Short fragments of DNA containing.... Primers attach to sites on the DNA strands that are at either end of the segment you want to copy. They are powerful tools for copying very specific DNA sequeces since there is almost no chance that they will target the wrong sites. Taq Polymerase: Polymerase molecules read the DNA code then attach matching nucleotides to create DNA copies. DNTP's: nucleoside triphosphates containing deoxyribose. DNTP's are the building blocks for DNA. Q2: initial step(95 deg for 3 min): Begins to heat up and the bonds start to loosen denature(95 for 30 sec): The DNA double helix separates, creating two single-stranded DNA molecules. Anneal(57 for 30 sec): The single-stranded DNA molecules naturally attempt to pair up. Extend (72 for 30 sec): The DNA polymerase is activated. When DNA polymerase locates a primer attached to a single DNA strand, it begins to add complementary nucleotides onto the srtand. It continues until it gets to the end of strand and falls off. final Step (72 for 3 min): Holds the temperature and ends the cycle. Then it continues to do 29 more cycles, to produce over a billion fragments that contain only the target sequence and only sixty copies of the longer length molecules. Final Hold(4):