- 1 Growing a Bacterial E. coli Culture
- 2 Plasmid Minipreps: Qiagen Miniprep Kit
- 3 DNA Digests
- 4 DNA Gel Electrophoresis and Fragment Purification
- 5 Measuring DNA Concentration using a Nanodrop Spectrophotometer
- 6 Ligation Reaction
- 7 Transformation of E. coli
- 8 Confirming the Assembly
- 9 Growing Arabidopsis
- 10 Growing Agrobacterium
- 11 Making Agrobacterium Electro-Competent Cells
- 12 Arabidopsis transformation via Flower Dipping
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.
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
Measuring DNA Concentration using a Nanodrop Spectrophotometer
Transformation of E. coli
Confirming the Assembly
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!