IGEM:Harvard/2010/General Protocols

Growing a Bacterial E. coli Culture

E. coli is a typical bacteria used for molecular cloning. Long-term storage of BioBrick plasmids is in E. coli suspended in glycerol (500ul cells:500ul glycerol) and kept at -80˚C. Growing E. coli is performed at 37˚C, at which temperature they divide every 20 minutes.

• Day1: Making streaks from glycerol stocks
  • Warm agar plate at 37˚C for 10 minutes
  • Label lid of plate with what you're streaking on it, and your name, and the date
  • Locate desired glycerol stock in -80˚C freezer and, using a sterile toothpick, scrape out a tiny bit and streak on plate
  • Incubate plate overnite at 37˚C

• Day2: Growing liquid cultures
  • Label 15mL falcon tube and add 5mL of LB media + selective antibiotic
  • Using 200uL tip w/o filter, touch streak or single colony and put tip in falcon tube
  • Grow culture overnite in a shaking 37˚C incubator

Plasmid Minipreps: Qiagen Miniprep Kit

To extract plasmid DNA from bacteria, perform a miniprep using the Qiagen miniprep protocol.

DNA Digests

Use appropriate enzyme to cut the BioBrick plasmids or parts to be ligated, taking care as to whether you're performing a "front-end" or "back-end" ligation.

• Write out exactly what you're building, i.e. part names, sizes, etc

• In a 1.5mL centrifuge tube, set up digest reaction as follows:

• • Plasmid DNA: ____ uL = ~1000ng
  • Enzyme 1: 1.0 uL
  • Enzyme 2: 1.0 uL
  • Buffer: 2.0 uL
  • Water: ____ uL

____________________________________

• • Total: 20.0 uL

(Note: Always be sure that the TOTAL enzyme concentration is never >10% of TOTAL volume, i.e. 2uL enzymes/20uL total reaction is 10%.)

• Mix tube well and digest at 37˚C for at least 5 minutes. If you are including a phosphotase enzyme, you need to deactivate it by next incubating the tube at 65˚C for 10 minutes.

• Proceed to gel purification.

DNA Gel Electrophoresis and Fragment Purification

• • • • • NOTE: Ethidium Bromide (EtBr) is a mutagen ---- always wear gloves when handling it*****
• Making an agarose gel
  • To make a 1% gel, add 1g agarose to 100mL 1x TAE buffer in 200mL flask
  • Microwave for 1.5 minutes
  • Swirl and check that the agarose is completely dissolved and there are no air bubbles
  • Allow gel solution to cool for a bit
  • While gel is cooling, tape the ends of gel mold so gel liquid cannot escape. Don't forget the combs!! Sit mold on a flat surface.
  • Add 4.5ul of EtBr to 100mL of gel and mix by swirling (avoid making bubbles)
  • Pour into taped gel mold and allow to cool until solid

Growing Agrobacterium

We have received from Amy Hark the agrobacterium strain: GV3101 pMP90. This is the strain that was used to transform the plant vectors we purchased into Arabidopsis.

• Frequently used for many binary vectors for Arabidopsis
• Resistance to Rif (10mg/L) (in genome), Gent (30mg/L) (in helper plasmid), and possibly Kan (30mg/L) (in helper plasmid) ---- she told me she usually only selects with Gent, but we may want to try and see if it is still resistant to Kan
• Known to develop spontaneous Tet resistance, so this strain should be used with care.
• Fast growing colonies after transformation are false. True transformants appear 1-2 days after fast growing colonies.
• Use 100ug/mL timentin/cefotaxim for Agro removal when selecting transgenic plants on plate
• Use bromin to kill agro.

Making Agrobacterium Electro-Competent Cells

This protocol should be doubled up - it is almost the same amount of work and you can get some 80 tubes.

• Inoculate colony O/N in 3mL LB+antibiotics at 30˚C shaker
• Transfer O/N cultures to 200mL LB in sterile 500mL flask and shake at 250rpm until the OD is 0.3 (4-5h)
• Spin in sterile screw cap tubes at 4˚C, 5000 rpm for 10min. Check to make sure cells are pelleted - if not repeat at higher speed
• Aspirate supernatant, resuspend pellet in 20mL ice cold 1mM HEPES pH7 (sterile filtered), re-spin. Repeat this step 2 more times.
• After aspirating, resuspend pellet in 2mL ice cold 10% glycerol (sterile filtered)
• ASAP dispense in 40ul aliquots in pre-chilled, sterile eppis, freeze in liquid nitrogen and store at -70˚C

Arabidopsis transformation via Flower Dipping

• Grow 3mL O/N cultures of construct in AgroGV3101 with proper antibiotics selection
• Inoculate into 250mL LB kan/gent (does 4-6 pots)
• Spin down, 7k X 5min
• Resuspend in 5% sucrose, 0.05% Silwet (50g sucrose, 0.5mL Silwet, 1L distilled water)
• Dunk plants in the bacterial suspension in a 1L beaker for 1-2 sec
• Lay pots on their sides in a flat and cover with saran wrap and leave O/N in growth chambers
• Take off saran wrap and put upright. Clean tray if too messy - can get smelly!