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| =Growing a Bacterial E. coli Culture= | | =Synthetic Biology= |
| 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.
| | *[[IGEM:Harvard/2010/General Protocols/BioBricks | How Do BioBricks Work?]] |
| | *[[Silver: BB Strategy | Strategy for Making Parts]] |
| | *[[Silver: PCR | PCR]] |
| | *[[Silver: Oligonucleotide Inserts | Oligonucleotide Inserts]] |
| | *[[Silver: Site-Directed Mutagenesis | Site-Directed Mutagenesis]] |
| | *[[Silver: Restriction Digest | Restriction Digest]] |
| | *[[IGEM:Harvard/2010/General Protocols/DNApurification | DNA Gel Electrophoresis and Fragment Purification]] |
| | *[[IGEM:Harvard/2010/General Protocols/Nanodrop | Measuring DNA Concentration Using a Nanodrop Spectrophotometer]] |
| | *[[Silver: Ligation | Ligation]] |
| | *[[Silver: Quick & Dirty Digest & Ligation | Quick & Dirty Digest & Ligation]] |
| | *[[Silver: Bacterial Transformation | Bacterial Transformation]] |
| | *[[Silver: Plasmid Verification | Plasmid Verification]] |
| | *[http://labs.fhcrc.org/fero/Protocols/RNeasy_Mini_Handbook.pdf Qiagen RNeasy Plant Mini Kit] |
| | *[http://cshprotocols.cshlp.org/cgi/reprint/2007/4/pdb.kit23.pdf iScript cDNA Synthesis] |
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| *Day1: Making streaks from glycerol stocks | | =Microbiology= |
| **Warm agar plate at 37˚C for 10 minutes | | *[[IGEM:Harvard/2010/General Protocols/EColi | Growing E. coli]] |
| **Label lid of plate with what you're streaking on it, and your name, and the date | | *[[IGEM:Harvard/2010/General Protocols/Agrobacterium | Growing Agrobacterium]] |
| **Locate desired glycerol stock in -80˚C freezer and, using a sterile toothpick, scrape out a tiny bit and streak on plate
| | *[[IGEM:Harvard/2010/General Protocols/CompetentAgrobacterium | Making Electro-Competent Agrobacterium]] |
| **Incubate plate overnite at 37˚C | | *[[IGEM:Harvard/2010/General Protocols/Agrobacterium Electroporation | Electroporating Agrobacterium]] |
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| *Day2: Growing liquid cultures
| | =Plants= |
| **Label 15mL falcon tube and add 5mL of LB media + selective antibiotic
| | *[[IGEM:Harvard/2010/General Protocols/Arabidopsis | Growing Arabidopsis]] |
| **Using 200uL tip w/o filter, touch streak or single colony and put tip in falcon tube
| | *[[IGEM:Harvard/2010/General Protocols/FlowerDipping | Arabidopsis transformation via Flower Dipping]] |
| **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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| ____________________________________
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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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| =Growing Agrobacterium=
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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 | |
| *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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| *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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