1 iGEM 2018 Lab Book 1 2 How to use this book Welcome to the iGEM 2018 Lab Book. Here, we will record all our experiments, failures and success alike. This is our story. Our adventures begin now... But first, here are a few guidelines for using this online lab book. Refer to your personal lab book Every lab team member has a personal physical lab book. Here, we’re transfering all of our experiments into a signal lab book. All experiments should be recorded in an accurate, detailed, and complete manner in both personal and shared lab books. When transfering things to this book, be sure to include the include a reference to your lab book. Do this by including “Refer to your first and last name’s lab book, p.#” at the top left corner of the ​ ​ ​ ​ ​ ​ ​ page, after the title of your experiment and date (see template). This will help keep things organized and allow us to check the original lab book and catch any transcription errors. Use sufficiently descriptive titles Instead of a table of contents, we’re gonna use the Outline feature of Google Docs. Be sure to write titles in “Heading 2” and the rest of your experiments in “normal text”. To make ​ ​ ​ ​ it easy to refer back to each experiment, give each one a descriptive title. Don’t just call it “PCR”, but rather “PCR of thing you’re trying to amplify - attempt 1”. For more detail about ​ ​ this, refer to templates of different experiments. Follow given templates To keep entries consistent, we’ve created templates for different experiments (PCR, digests, heat-shock, etc.). These can be found in the following pages. Simply copy, paste, and fill in the blanks. 2 3 Templates General template for all entries: Micro Team (write “other” if none) - Title of experiment Your full name Date Performed “Refer to your first and last name’s lab book, p.#” ​ ​ ​ ​ ​ If you followed the protocols in the protocol section of this book: “Followed experimental procedure performed protocol without any deviations” ​ ​ ​ If you followed the protocols in the protocol section of this book, but made a few changes: “Followed experimental procedure performed protocol with the following deviations: ​ ​ ​ ● List deviations you made ● ...” [..............................................................Your results……………………………………………] Protocols Inoculation 1. Aseptically pipette 5 mL sterile liquid broth into a test tube. 2. Add appropriate antibiotic, if required, to desired concentration using a pipette. Volumes depend on antibiotic but there is a list stuck onto the -20℃ freezer in the iGEM lab. 3. When pipetting the antibiotic, tilt the test tube so that the broth is close enough to the top of the tube that you can add the antibiotic to the broth. Dropping it in won’t work because you will typically be adding ~ 2.5 μL and the drop will be too small to fall. 4. If inoculating from frozen stock, work quickly! This is very important because if ​ ​ the cells thaw completely they will have a shorter lifetime in the freezer. Always keep 3 4 frozen stocks on ice. 6. Take an autoclaved wooden inoculation stick and scrape some frozen stock from tube and dip the stick into liquid media and stir around. 7. Discard inoculation stick into another container, not the waste! Inoculation sticks are reusable. 8. If inoculating from a plate. Take a wooden stick and touch a colony on the plate ​ ​ then dip it into the liquid media and stir around. DON’T discard sticks. 9. Place the tubes in a 37℃ incubator on the shaker for 12-24 hours. If possible, angle the tubes because it improves oxygenation of the culture as it shakes. Nanodrop The NanoDrop (ND-1000) is a small-scale spectrophotometer that can detect DNA concentration and give an estimate of sample purity. 1. Go to the“core facility” across from the elevator in B1. The door code is 933214 ​ 2. Open the nanodrop application on the computer and login to the iGEM account using the password “bio”. ​ ​ 3. Click on “Nucleic acid” then begin to initialize the unit. ​ ​ 4. Put some milliQ water on a KimWipe and wipe down the pedestal and sampling arm. 5. Add 1.0 μL of ddH2O, then 1.0μL of elution buffer to blank. ​ ​ 6. Load 1.0 μL of sample and click “Measure” to determine the concentration and absorbance ratios. Record these below. Between trials, wipe the machine with a kimwipe. Make sure to leave machine how you found it Plasmid miniprep 1. Pellet 1–5 ml bacterial culture (not to exceed 15 OD units) by centrifugation for 30 seconds. Discard supernatant. 2. Resuspend pellet in 200 μl Plasmid Resuspension Buffer (B1) (pink). Vortex or pipet ​ ​ to ensure cells are completely resuspended. There should be no visible clumps. 3. Lyse cells by adding 200 μl Plasmid Lysis Buffer (B2) (blue/green). Invert tube ​ ​ immediately and gently 5–6 times until color changes to dark pink and the solution is clear and viscous. Do not vortex! Incubate for one minute. 4 5 4. Neutralize the lysate by adding 400 μl of Plasmid Neutralization Buffer (B3) (yellow). ​ ​ Gently invert tube until color is uniformly yellow and a precipitate forms. Do not vortex! Incubate for 2 minutes. 5. Clarify the lysate by spinning for 2–5 minutes at 16,000 x g. 6. Carefully transfer supernatant to the spin column and centrifuge for 1 minute. Discard flow-through. 7. Re-insert column in the collection tube and add 200 μl of Plasmid Wash Buffer 1. Plasmid Wash Buffer 1 removes RNA, protein and endotoxin. Centrifuge for 1 minute. Discarding the flow-through is optional. 8. Add 400 μl of Plasmid Wash Buffer 2 and centrifuge for 1 minute. 9. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column has not come into contact with the flow-through. If there is any doubt, re-spin the column for 1 minute before inserting it into the clean microfuge tube. 10. Add ≥ 30 μl DNA Elution Buffer to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA. PCR Recipe: Reagent Per reaction (in uL) Jumbo cocktail for __ rxns (in uL) Forward Primer (10 μM) 1 Reverse Primer (10 μM) 1 Taq/Q5/Phusion Master Mix 10 Sterile, nuclease-free water 8 Add template individually to each sample. Template may be a few cells (i.e. picked colony, small volume of liquid culture), or extracted/purified DNA (i.e. plasmid, genome, linear). Typically, add 1uL of ~1ng/uL extracted/purified DNA (dilute in nuclease-free water if needed) or 1uL of overnight culture. For colony PCR, simply touch the colony with a sterile tip and mix that into your reaction tube. Thermocycler procedure: Q5 2X HF MM Taq MM Phusion MM Step Temp time Temp time Temp time (in C) (in C) (in C) Initial 98 30 sec 95 30 sec 98 30 sec denaturation denaturation 98 5-10 sec 95 15-30 sec 98 5-10 sec 5 6 annealing 50-72 * 10-30 sec 45-68 * 15-60 sec 45-72 * 10-30 sec extension 72 20-30 sec/kb 68 1 min/kb 72 15-30 sec/kb Final 72 2 min 68 5 min 72 5-10 min extension hold 4 Infinity 4 Infinity 4 Infinity * annealing temperature depends on the primers you’re using. Note that this is for standard PCR (where your template is extracted DNA). If your template is DNA in cells (i.e. colony PCR), then initial denaturation should be 5-10 min to lyse the cells. Gel electrophoresis Setting up your rig: 1. Usually we use the small rig with the corresponding small tray. 2. Tape the edges of the tray. This needs to be water-tight, as you will set your gel here. Choosing a comb: There are several reasons you might want to run a gel, which correspond to different choices of comb. 1. If you are running a gel to visualize a result, use the comb with the smallest teeth, and use the side with the thinnest teeth. Usually you run a small amount of sample in a gel like this, so the wells don’t need to be large. 2. If you are running a gel for the purposes of gel extraction, use the comb with the largest teeth, and use the side with the thickest teeth. Usually you run a large amount of sample in a gel like this, so the wells need to be large. Pouring and running your gel: 1. Add agarose into a flask to make the concentration of agarose 0.8-1.2% (e.g. 0.5 g agarose to make 50 mL 1% agarose gel) 2. Add desired amount of 1X TAE running buffer (50 mL for small tray use more if you are running a larger gel). 6 7 3. Microwave 30 seconds. Using rubber mitt, vigorously swirl the partially-dissolved agarose in the flask. 4. Microwave 15 seconds. Using rubber mitt, vigorously swirl the dissolved agarose in the flask. Visually inspect the solution to make sure there is no undissolved agarose floating in it. Keep microwaving (in short intervals) until fully dissolved. 5. Add Gel Red at 1:10 000 concentration (50 mL gel needs 5 μL gel red). 6. Pour gel into tray. 7. Put comb into place 8. Let it sit for 20 minutes - until gel loses some transparency and looks more “white”. 9. Once the gel has set, remove the tray from the rig and replace it in the rig so the ends of the tray are open.