Difference between revisions of "IGEM:Harvard/2010/General Protocols"

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(Making Agrobacterium Electro-Competent Cells)
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=Growing a Bacterial E. coli Culture=
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=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.
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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]
  
*Day1: Making streaks from glycerol stocks
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=Microbiology=
**Warm agar plate at 37˚C for 10 minutes
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*[[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
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*[[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
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*[[IGEM:Harvard/2010/General Protocols/CompetentAgrobacterium | Making Electro-Competent Agrobacterium]]
**Incubate plate overnite at 37˚C
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*[[IGEM:Harvard/2010/General Protocols/Agrobacterium Electroporation | Electroporating Agrobacterium]]
  
*Day2: Growing liquid cultures
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=Plants=
**Label 15mL falcon tube and add 5mL of LB media + selective antibiotic
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*[[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
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*[[IGEM:Harvard/2010/General Protocols/FlowerDipping | Arabidopsis transformation via Flower Dipping]]
**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
 
 
 
=Measuring DNA Concentration using a Nanodrop Spectrophotometer=
 
=Ligation Reaction=
 
=Transformation of E. coli=
 
=Confirming the Assembly=
 
=Growing Arabidopsis=
 
=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!
 

Latest revision as of 10:54, 1 July 2010

Synthetic Biology

Microbiology

Plants