"

From 2011.igem.org

DNA KIT PLATE INSTRUCTIONS Materials Transformation

   Equipment

• Incubator

• Water bath

• Petri dishes (with LB agar and appropriate antibiotic/ two per a transformation)

• Pipette tips

• Micropipettes

• Sterile tubes

• Sterile loops

• Racks

    Reagents 

• LB agar

• SOC (Check for contamination; see preparation in annexes)

• Distilled water

• Antibiotics (according to each BioBrick)

    Biological agents 

• Resuspended DNA

• Competent cells

Miniprep plasmid DNA isolation

  Equipment 

• Centrifuge

• Eppendorf tubes

• Vortex

• Magnetic stirrer (for boiling water)

  Reagents 

• Buffers o STET  8% sucrose  50mM Tris-HCl, pH 8  0.5% Triton X-100  50mM EDTA (ethylene-diamine-tetraacetic acid) o TE  10mM Tris-HCl, pH 8  1mM EDTA • Ice cold isopropanol • Ice cold 80% ethanol

  Biological agents 

• Lysozyme • Plasmids (in overnight cultures)

DNA glycerol stock • 40% glycerol solution • Cryogenic vials General Protocol To use the DNA in the Distribution Kit you may follow these instructions: 1. With a pipette tip, punch a hole through the foil cover into the corresponding well of the Biobrick™-standard part that you want. Make sure you have properly oriented the plate. We recommend that you do not remove the foil cover, as it could lead to cross contamination between the wells. But it is important to take care of the plate orientation after punching the foil. 2. Pipette 10uL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. We recommend that you do not use TE to resuspend the dried DNA. 3. Transform 1 or 2uL of the resuspended DNA into your desired competent cells, plate your transformation with the appropriate antibiotic and grow overnight.

Bacterial Transformation

a) Start thawing the competent cells on crushed ice.

b) Add 50 µL of thawed competent cells and then 1 - 2 µL of the resuspended DNA to the labelled tubes. Make sure to keep the competent cells on ice.

c) Incubate the cells on ice for 30 minutes.

d) Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds. A water bath improves heat transfer to the cells.

e) Incubate the cells on ice for 5 minutes.

f) Add 200 μl of SOC broth (make sure that the broth does not contain antibiotics and is not contaminated)

g) Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. Important: 2 hour recovery time helps in transformation efficiency, especially for plasmids with antibiotic resistance other than ampicillin.

h) Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid, and antibiotic resistance. Plate 20 µl and 200 µl of the transformation onto the dishes, and spread. This helps ensure that you will be able to pick out a single colony.

i) Incubate the plate at 37ºC for 12-14 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics start to break down and un-transformed cells will begin to grow. This is especially true for ampicillin - because the resistance enzyme is excreted by the bacteria, and inactivate the antibiotic outside of the bacteria.

4. Pick a single colony and inoculate broth (with the correct antibiotic) and grow for 16 hours.

5. Use the resulting culture to miniprep.

       Miniprep Plasmid DNA Isolation

a) Transfer 1.5 mL of an overnight culture containing your plasmid to an eppendorf tube and spin at 5000 rpm for 5 min in a tabletop centrifuge to pellet the cells.

b) Remove and discard the supernatant.

c) Add 300 μL STET buffer, and resuspend cells by vortexing.

d) Add 10 μL lysozyme (10 mg/mL), vortex, and submerse in boiling water for 40 sec.

e) Spin for 30 min in a tabletop centrifuge at maximum speed at 4 ˚C.

f) Remove pellet from each tube with a toothpick. The cellular debris should stick well to the toothpick or platinum loop. Try to insert and remove the toothpick from the center of the tube so you don't get any cellular debris on the sides of the tube.

g) Add 300 μL ice cold isopropanol to precipitate the DNA (or 300μL of 2:1 isopropanol: ammonium acetate, mixed just before you use it.)

h) Spin for 10 min in a tabletop centrifuge at maximum speed at 4 ˚C.

i) Remove supernatant.

j) Add 200 μL ice cold 80% ethanol to wash pellet and spin for 5 min in a tabletop centrifuge at maximum speed at 4 ˚C.

k) Remove supernatant.

l) Dry pellet (air dry at room temperature or 37 ˚C or dry in a speedvac).

m) Rehydrate in 50 μL TE.

Glycerol stock

a) Add 1 ml of 40% glycerol in H2O to a cryogenic vial.

b) Add 1 ml sample from the culture of bacteria to be stored.

c) Gently vortex the cryogenic vial to ensure the culture and glycerol is well-mixed. a. Alternatively, pipet to mix

d) Use a tough spot to put the name of the strain or some useful identifier on the top of the vial.

e) On the side of the vial list all relevant information - part, vector, strain, date, researcher, etc.

f) Store in a freezer box in a -80C freezer. Remember to record where the vial is stored for fast retrieval later.