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Team:Virginia/Protocols
From 2008.igem.org
Contents |
QIAGEN Mini-Prep
- Spin cell broth down in centrifuge at >13200 revolutions per minute in microcentrifuge (generally 4 minutes is adequate).
- Pour off remaining broth and resuspend in 250 uL of Buffer P1 (LyseBlue is nice to have).
- Add 250 uL Buffer P2 (for cell lysis). Invert microcentrifuge tubes 4-6 times. Do NOT allow to run for more than 5 minutes.
- Add 350 uL Buffer N3 and invert tubes another 4-6 times.
- Spin in microcentrifuge at >13000 revolutions per minute for ten minutes.
- Transfer supernatant to provided spin column.
- Run spin column in microcentrifuge for 60 seconds. Discard flow-through.
- Add 0.5 mL Buffer PB. Centrifuge for 60 seconds. Discard flow-through.
- Add 0.75 mL Buffer PE. Centrifuge for 60 seconds. Discard flow through.
- Centrifuge for an additional 60 seconds.
- Transfer spin-column top to a microcentrifuge tube for collection.
- Add 50 uL Buffer EB to center of column. Allow to sit for one minute.
- Centrifuge for one minute. The clear liquid in the bottom is your DNA solution.
Gel Electrophoresis (Making the gel)
- In a 100 mL Erlenmeyer flask, place and pour: 0.5 g Agar and 50 mL 1X TAE Buffer
- Microwave for 30 seconds and then swirl the flask
- Microwave for 15 seconds (caution: do not let the solution boil out of the flask)
- Pipette 2 microliters of Ethidium Bromide into the gel solution and then swirl flask
- Ethidium Bromide stains the plate for DNA to be observed under UV light
- NOTE: Ethidium Bromide is a hazardous chemical and must be disposed of properly - this includes the gel itself
- Setup gel holder with desired size and number comb perpendicular leads so that liquid gel stays contained
- Pour all of hot gel into gel holder, cover with saran wrap and leave out to solidify
- This is a 1% agar gel; depending on the size of your DNA fragment, you will want to increase concentration of the agar for smaller pieces of DNA
- for DNA fragments between 10 to 100 basepairs - it is advisable you use specially prepared agar such as nusieve GTG agar which can be obtained online
Gel Electrophoresis (Loading & Running)
- Align wells on the side of the negetive(black) lead
- Fill with 1X TAE buffer to cover gel with thin layer of buffer
- carefully remove the comb
- Using small epindorf tubes mix together DNA and 6X loading dye with a 6:1 ratio (this ratio is dependant on the loading dye)
- Mix 1 uL of 1kb ladder with 5 uL of water and 1 uL of 6X loading maintaining the ratio
- Carefully pipette the DNA mixtures into the wells being careful not to pierce the bottom of the wells
3A assembly Ligation
- Assuming the 3 parts that are going to be ligated are cut appropriately
- First part cut at EcoRI and SpeI
- Second part cut at XbaI and PstI
- Back bone/Vector cut at EcoRI and PstI
- this works because S and X form a scar and the other restriction sites are preserved and the ligated insert keeps the same format with EX as a prefix and SP as a suffix
- the back bones should have a different antibiotic resistance than the 2 inserts to guarantee that any colonies that grow are products of a successful ligation
- Note: should not be done in DB3.1 cells
- For 20ul mix:
- 4 ul water
- 2 ul T4 10X ligase buffer
- 6 ul insert 1
- 6 ul insert 2
- 1 ul vector
- 1 ul T4 DNA ligase (enzyme)
- For 50ul mix:
- 17 ul water
- 5 ul T4 10X ligase buffer
- 12 ul insert 1
- 12 ul insert 2
- 2 ul vector
- 2 ul T4 DNA ligase (enzyme)
DNA Elution
- Obtain Parts binder and confirm desired Plate and Well number
- Warm a 10µL aliquot of 10:1 TE Buffer pH 8.0 in 1.5mL labeled microcentrifuge tube in the dry heating block to 50°C for each desired part
- Clean punch tool
- Punch clean thesis paper (remember cutting pad) and discard resulting chad
- Disassemble punch tool into pushrod, casing, and endcap (spring, endcap chamber, endcap lid)
- Dip pushrod and blade into each stage of cleaning station in sequence: bleach, distilled water, distilled water, 190 proof ethanol
- Blot clean with Kimwipe
- Let air dry on Kimwipe for 5 minutes
- Reassemble punch tool
- Confirm Plate and Well number
- Punch part
- Ensure punch tool is completely dry by tapping out on a Kimwipe!
- Slide cutting pad (in back of binder) underneath the binder page with your part
- Push (hard!) the punch tool blade into the corner of the desired filter paper stain, rotating to cut
- Once filter paper is cut, use pusher end of punch tool to eject chad into the corresponding labeled tube of TE Buffer
- Clean punch tool and repeat for each desired part
- Let sit for 20 minutes at 50°C in the dry heating block
- Centrifuge at 13,000 rpm for 3 minutes to maximize DNA concentration in solution
- Keep unused eluted DNA in 4°C fridge
Cell Transformation
- If retrieving previously eluted DNA from the fridge, let sit 20 minutes at 50°C in dry heating block
- Obtain bucket of ice
- Retrieve competent cells from -80°C freezer and place immediately on ice
- Transform the cells
- Label a 1.5mL microcentrifuge tube for each desired transformation and place on ice
- Pipette 25μL of thawed cells into each tube
- Pipette 5μL eluted DNA into each tube, inserting under the surface of the cells
- Let sit on ice for 30 minutes (put unused cells and DNA back)
- Heat shock in 42°C water bath for 1 minute if DH5α, 45 seconds if DB3.1
- Let sit on ice for 2 minutes
- Add 225μL of SOC Medium (check for turbidity before use) to each tube
- Place in agitating incubator and let shake at 37°C for 2 hours
- Remove plates with corresponding antibiotics from fridge well in advance of plating so they're room temperature
- Plate each tube of cells
- Dip glass spreader in 95% ethanol and burn on Bunsen burner, giving plenty of time to cool completely
- Pipette 200μL from each tube onto labeled plate
- Spread over surface of plate with sterile glass spreader
- Resterilize spreader and repeat with the rest of the tubes
- Place the plates LB down in 37° incubator (not shaking) for 5 minutes
- Turn the plates over (to ensure condensation doesn't kill the cells) and incubate overnight
