IGEM:Harvard/2010/General Protocols

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Arabidopsis transformation via Flower Dipping)
(Growing an Agrobacterium Culture)
Line 53: Line 53:
=Confirming the Assembly=
=Confirming the Assembly=
=Growing Arabidopsis=
=Growing Arabidopsis=
-
=Growing an Agrobacterium Culture=
+
=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)
 +
7. ASAP dispense in 40ul aliquots in pre-chilled, sterile eppis, freeze in liquid nitrogen and store at -70˚C
 +
 
=Arabidopsis transformation via Flower Dipping=
=Arabidopsis transformation via Flower Dipping=
*Grow 3mL O/N cultures of construct in AgroGV3101 with proper antibiotics selection
*Grow 3mL O/N cultures of construct in AgroGV3101 with proper antibiotics selection

Revision as of 11:21, 3 June 2010

Contents

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

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)

7. 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!
Personal tools