IGEM:Harvard/2010/General Protocols

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(Growing an Agrobacterium Culture)
Current revision (13:54, 1 July 2010) (view source)
(Microbiology)
 
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=Growing a Bacterial E. coli Culture=
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=Synthetic Biology=
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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.
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*[[IGEM:Harvard/2010/General Protocols/BioBricks | How Do BioBricks Work?]]
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*[[Silver: BB Strategy | Strategy for Making Parts]]
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*[[Silver: PCR | PCR]]
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*[[Silver: Oligonucleotide Inserts | Oligonucleotide Inserts]]
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*[[Silver: Site-Directed Mutagenesis | Site-Directed Mutagenesis]]
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*[[Silver: Restriction Digest | Restriction Digest]]
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*[[IGEM:Harvard/2010/General Protocols/DNApurification | DNA Gel Electrophoresis and Fragment Purification]]
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*[[IGEM:Harvard/2010/General Protocols/Nanodrop | Measuring DNA Concentration Using a Nanodrop Spectrophotometer]]
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*[[Silver: Ligation | Ligation]]
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*[[Silver: Quick & Dirty Digest & Ligation | Quick & Dirty Digest & Ligation]]
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*[[Silver: Bacterial Transformation | Bacterial Transformation]]
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*[[Silver: Plasmid Verification | Plasmid Verification]]
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*[http://labs.fhcrc.org/fero/Protocols/RNeasy_Mini_Handbook.pdf Qiagen RNeasy Plant Mini Kit]
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*[http://cshprotocols.cshlp.org/cgi/reprint/2007/4/pdb.kit23.pdf iScript cDNA Synthesis]
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*Day1: Making streaks from glycerol stocks
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=Microbiology=
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**Warm agar plate at 37˚C for 10 minutes
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*[[IGEM:Harvard/2010/General Protocols/EColi | Growing E. coli]]
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**Label lid of plate with what you're streaking on it, and your name, and the date
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*[[IGEM:Harvard/2010/General Protocols/Agrobacterium | Growing Agrobacterium]]
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**Locate desired glycerol stock in -80˚C freezer and, using a sterile toothpick, scrape out a tiny bit and streak on plate
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*[[IGEM:Harvard/2010/General Protocols/CompetentAgrobacterium | Making Electro-Competent Agrobacterium]]
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**Incubate plate overnite at 37˚C
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*[[IGEM:Harvard/2010/General Protocols/Agrobacterium Electroporation | Electroporating Agrobacterium]]
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*Day2: Growing liquid cultures
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=Plants=
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**Label 15mL falcon tube and add 5mL of LB media + selective antibiotic
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*[[IGEM:Harvard/2010/General Protocols/Arabidopsis | Growing Arabidopsis]]
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**Using 200uL tip w/o filter, touch streak or single colony and put tip in falcon tube
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*[[IGEM:Harvard/2010/General Protocols/FlowerDipping | Arabidopsis transformation via Flower Dipping]]
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**Grow culture overnite in a shaking 37˚C incubator
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=Plasmid Minipreps: Qiagen Miniprep Kit=
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To extract plasmid DNA from bacteria, perform a miniprep using the Qiagen miniprep protocol.
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=DNA Digests=
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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.
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*Write out exactly what you're building, i.e. part names, sizes, etc
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*In a 1.5mL centrifuge tube, set up digest reaction as follows:
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**Plasmid DNA:      ____    uL = ~1000ng
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**Enzyme 1:            1.0      uL
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**Enzyme 2:            1.0      uL
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**Buffer:                  2.0      uL
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**Water:                  ____    uL
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**Total:                    20.0  uL
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(Note: Always be sure that the TOTAL enzyme concentration is never >10% of TOTAL volume, i.e. 2uL enzymes/20uL total reaction is 10%.)
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*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.
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*Proceed to gel purification.
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=DNA Gel Electrophoresis and Fragment Purification=
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*****NOTE: Ethidium Bromide (EtBr) is a mutagen ---- always wear gloves when handling it*****
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*Making an agarose gel
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**To make a 1% gel, add 1g agarose to 100mL 1x TAE buffer in 200mL flask
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**Microwave for 1.5 minutes
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**Swirl and check that the agarose is completely dissolved and there are no air bubbles
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**Allow gel solution to cool for a bit
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**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.
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**Add 4.5ul of EtBr to 100mL of gel and mix by swirling (avoid making bubbles)
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**Pour into taped gel mold and allow to cool until solid
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=Measuring DNA Concentration using a Nanodrop Spectrophotometer=
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=Ligation Reaction=
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=Transformation of E. coli=
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=Confirming the Assembly=
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=Growing Arabidopsis=
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=Making Agrobacterium Electro-Competent Cells=
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This protocol should be doubled up - it is almost the same amount of work and you can get some 80 tubes.
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*Inoculate colony O/N in 3mL LB+antibiotics at 30˚C shaker
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*Transfer O/N cultures to 200mL LB in sterile 500mL flask and shake at 250rpm until the OD is 0.3 (4-5h)
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*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
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*Aspirate supernatant, resuspend pellet in 20mL ice cold 1mM HEPES pH7 (sterile filtered), re-spin. Repeat this step 2 more times.
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*After aspirating, resuspend pellet in 2mL ice cold 10% glycerol (sterile filtered)
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7. ASAP dispense in 40ul aliquots in pre-chilled, sterile eppis, freeze in liquid nitrogen and store at -70˚C
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=Arabidopsis transformation via Flower Dipping=
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*Grow 3mL O/N cultures of construct in AgroGV3101 with proper antibiotics selection
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*Inoculate into 250mL LB kan/gent (does 4-6 pots)
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*Spin down, 7k X 5min
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*Resuspend in 5% sucrose, 0.05% Silwet (50g sucrose, 0.5mL Silwet, 1L distilled water)
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*Dunk plants in the bacterial suspension in a 1L beaker for 1-2 sec
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*Lay pots on their sides in a flat and cover with saran wrap and leave O/N in growth chambers
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*Take off saran wrap and put upright. Clean tray if too messy - can get smelly!
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Microbiology

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