Team:Virginia/Protocols

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

Stock Solutions of Antibiotics

 * For Ampicillin:
 * This will produce a 100mg/mL concentration
 * 0.5 grams Ampicillin
 * 2.5 mL of sterile water (can use MilliQ water or the like)
 * 2.5 mL of ethanol (100% pure)
 * Store in sterile eppendorf tubes and store in -20 degree fridge
 * For Kanamycin:
 * This will produce a 50mg/mL concentration
 * 100 mg of Kanamycin
 * 2 mL of sterile water (can use MilliQ water or the like)
 * Store in sterile eppendorf tubes and store in -20 degree fridge
 * For Chloramphenicol:
 * This will produce a 20 mg/mL concentration
 * 60 milligrams Chloramphenicol
 * 3 mL of Methanol
 * Store in sterile eppendorf tubes and store in -20 degree fridge
 * For Tetracycline:
 * This will give 10 mg/mL concentration
 * 20 mg of Tetracycline
 * 2 mL of sterile water (can use MilliQ water or the like)
 * Store in sterile eppendorf tubes and store in -20 degree fridge

LB Broth Solution

 * For making the LB broth mixture in powder form, in the following ratio of 10:5:5
 * 10 grams tryptone
 * 5 grams of yeast extract
 * 5 grams of NaCl
 * Mix 20 grams of LB broth mixture with 1 L of deionized water - use a magnetic stirring rod to ensure mixing
 * Cover flask with aluminum foil and tape top with autoclaving tape
 * Autoclave for 20 minutes in Liquid Cycle
 * NOTE: to add in antibiotic to the LB Broth Solution, you must wait until the LB broth has cooled after autoclaving (Flask must be cool enough to touch) otherwise the antibiotics will break down in the heat.

LB Broth Agar Plates

 * Prepare LB Broth Solution as stated above
 * after mixing 20 g with 1 L of deionized water, add 16 g of Agar to the flask
 * Use a magnetic stirring rod to ensure mixing
 * Cover flask with aluminum foil and tape top with autoclaving tape
 * Autoclave for 20 minutes in Liquid Cycle
 * NOTE: to add in antibiotic to the LB Broth Solution, you must wait until the LB broth has cooled after autoclaving (Flask must be cool enough to touch) otherwise the antibiotics will break down in the heat.
 * One liter of LB Agar Solution will roughly fill 25 plates; fill the plate to half full.
 * after the plates solidify, store in 4 degree Fridge with the top of the plates down (prevents condensation on the lid from dripping onto the gel)

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 25μL 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