Lab 3 A) Setting Up a PCR Reaction with Bacterial Cultures B) Start Plasmid DNA Miniprep

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1 Lab 3 A) Setting Up a PCR Reaction with Bacterial Cultures B) Start Plasmid DNA Miniprep A) Setting Up a PCR Reaction with Bacterial Cultures. (This protocol is set up for a group of four students each doing on clone apiece). 1. Each student in the group should first label a microfuge tube(s) the same name(s) as their bacterial culture (e.g. 20WS1.13). On the lid write DIL to indicate that this is your dilution tube. 2. Using a P200, add 200 µl of sterile ddh 2 O. 3. Resuspend the bacterial culture by lightly tapping the side of the tube to make sure that all the cells are suspended. 4. Take 5 µl of the overnight culture and add it to each corresponding labeled microfuge tube containing 200 µl of ddh 2 O. Mix each tube by tapping the tube. DO NOT DISCARD THE CULTURES!!! You will need them for your minipreps in part B. 5. Each group of four students should prepare a 5X primer mix i) Label an empty 1.7 ml microfuge tube with 5X Mix. ii) In this tube prepare a reaction digestion mixture as shown as shown below. This is the reaction mix for four clones (one clone for four students or four clones for one student). Prepare this same mixture even if each student is doing two clones each. 5X sterile ddh 2 O FOR Primer (SP) (10 pmole/µl) 90 µl 12.5 µl REV Primer (XP) (10 pmole/µl) 12.5 µl Total volume 115 µl Mix the ingredients of the PCR Mix by gently tapping the tube. 6. Each student should label one PCR tube(s) with a bead with an abbreviated clone name of the diluted colony sample that will be added to it (WS1, WS2, etc). The full clone name will not fit on the side of the tube WSSP 3-1

2 7. Each student should add 23 µl of the 5X Mix to their tube(s) containing a PCR Bead. 8. Each student should add 2 µl of the appropriately diluted culture that was prepared in Step 4 to the PCR tube. Mix each by gently tapping the tube. The bead should dissolve and produce foam. Discard the tubes with the diluted cultures. NOTE: The PCR beads provided this year are in 0.2 ml tubes. If your thermal cycler can use these tubes, then you proceed to the next step (9). If your thermal cycler can only use 0.5 ml tubes, then use a P-200 set at 25 µl to transfer the dissolved bead, template and primer mix from the 0.2 ml tube to a properly labeled 0.5 ml tube. DO NOT TRY TRANSFER THE BEADS FROM ONE TUBE TO ANOTHER 9. Add the PCR tubes to the thermal cycler. MAKE SURE THAT YOU HAVE LABELED THE SIDE OF YOUR TUBES WITH YOUR CLONE NAME. Write down the full clone name in the grid sheet for the thermal cycler in case the writing rubs off the tube. 10. Make sure the thermal cycler has the correct program, called WSSP : I Initial Denaturation 94 o C for 5 minutes (This insures that the cells are lysed and the DNA is thoroughly denatured.) II Amplification (The thermal cycler will repeat these three steps in order 30 times.) 94 o C for 30 seconds (Denature the target DNA) 52 o C for 30 seconds (Anneal the primer to template DNA) 72 o C for 1 minute (Elongate the primer to produce the new DNA strand) III Additional Elongation (This step insures all DNA strands are full length) 72 o C for 5 minutes IV End Program After step III, the thermal cycler has been programmed to drop the temperature of the heating block to 4 o C. Your samples will be very stable if left at 4 o C. If your thermal cycler program gets deleted or changed please see the Mastercycler Thermal Cycler Programing instructions posted on the web. 11. To start an Eppendorf MasterCycler thermal cycler: a) Place your PCR tubes into the thermal cycler. Little holes are for the 0.2 ml tubes and larger holes are for the 0.5 ml tubes. Use the Grid Template to write down the location of your tube WSSP 3-2

3 Lab 3: PCR on ONs b) Close the lid and lock it. c) Turn the thermal cycler ON by pressing the button located on the left back of the instrument. d) Use the down arrow key to move the cursor to Start, press the blue Enter button. There should only be a program called WSSP for a PCR amplification. Use that one! e) Press the blue Enter button to start the WSSP program. The complete amplification takes about 2.5 hours. f) When the PCR has finished, press the white Start/Stop button to end the program. A new screen will pop up. It will ask you if you want to abort the program. g) Press the blue Enter button to abort the program. This will end the program and bring you back to the original screen. h) Turn the thermal cycler OFF by pressing the button located on the left back of the machine. 12. After your PCR samples have run, store the PCR samples in the freezer until you are ready to run your agarose gel WSSP 3-3

4 Grid Template Machine # Lab 3: PCR on ONs 2013 WSSP 3-4

5 B) Start Plasmid DNA Miniprep See the minprep video on the WSSP site for the steps below. This protocol is based on each student performing one plasmid DNA miniprep 1. Each student in the group should first label one microfuge tube(s) the same name(s) as their cultures (e.g. 20WS1.13). 2. Gently tap the culture tube to resuspend the cells. 3. Pour 1.5 ml of each culture into the labeled microfuge tube and pellet the cells for 1 minute at full speed (14,000 rpm) in the microcentrifuge. 4. Remove the clear supernatant (sup) by pouring into a liquid waste container. 5. Resuspend the bacterial pellet in 200 µl of Solution I using your P1000 to disrupt the pellet and then gently pipeting up and down several times. Make sure that it is fully resuspended. 6. Store the tube in your school s box in the freezer (-20 C) and enter the tube s location in the box on the Google Docs Clone Report Sheet for your school WSSP 3-5