IGEM:Harvard/2010/General Protocols

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(DNA Digests)
(DNA Digests)
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*In a 1.5mL centrifuge tube, set up digest reaction as follows:
*In a 1.5mL centrifuge tube, set up digest reaction as follows:
-
**Plasmid DNA      ____ uL = ~1000ng
+
**Plasmid DNA:     ____     uL = ~1000ng
-
**Enzyme 1            1.0      uL
+
**Enzyme 1:           1.0      uL
-
**Enzyme 2            1.0      uL
+
**Enzyme 2:           1.0      uL
-
**Buffer                 2.0      uL
+
**Buffer:                  2.0      uL
-
**Water                  ____ uL  
+
**Water:                   ____     uL  
____________________________________
____________________________________
-
**Total                    20.0  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%.)
(Note: Always be sure that the TOTAL enzyme concentration is never >10% of TOTAL volume, i.e. 2uL enzymes/20uL total reaction is 10%.)

Revision as of 12:58, 17 May 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

Measuring DNA Concentration using a Nanodrop Spectrophotometer

Ligation Reaction

Transformation of E. coli

Confirming the Assembly

Growing Arabidopsis

Growing an Agrobacterium Culture

Arabidopsis transformation via Flower Dipping

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