Team:TzuChiU Formosa/Notebook

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Revision as of 03:34, 28 October 2010

Contents

Protocol

PCR (total volume: 50 μl )

  • 25 μl Green master mix (Promega)
  • 2 μl each primer
  • 2 μl template
  • 19 μl ddH2O
  • Cycle 95/2 min; 30x(95/1 min;56/1 min;72/2 min); 72/7 min

Gel Extraction- use Gel /PCR DNA Fragments Extraction Kit (Geneaid)

  • Loading the PCR products into agarose gel; run gel with 1X TAE buffer
  • Excise the agarose gel slice containing relevant DNA fregments
  • Transfer gel slice to a 1.5 ml microcentrifuge tube
  • Add 500 μl DF buffer; incubate at 56 ℃ until gel slice has been completely dissolved
  • Transfer 800 μl gel mixture to DF column
  • Centrifuge 13000 rpm, 1 min
  • Discard the flow-through and place the DF column back
  • Add 400 μl W1 buffer; Centrifuge 13000 rpm, 1 min; Discard the flow-through
  • Add 600 μl wash buffer; Centrifuge 13000 rpm, 1 min; Discard the flow-through
  • Centrifuge 13000 rpm, 5 min; air- dry
  • Add 50 μl ddH2O; Centrifuge 13000 rpm,1 min
  • Storage at – 20℃

TA Cloning- use pGEM®-T Easy Vector (Promega)

  • 1μl pGEM®-T Easy Vector
  • 5μl 2X rapid ligation buffer
  • 1μl T4 DNA ligase
  • 3μl insert
  • 4 ℃, overnight

Ligation on pSB1C3- use Elite T4 DNA ligase (Geneaid)

  • 9 μl insert
  • 3 μl vector
  • 2 μl 10X ligation buffer A
  • 2 μl 10X ligation buffer B
  • 1 μl Elite T4 DNA ligase
  • 3 μl ddH2O
  • 16℃, overnight

Transformation

  • Take out XL-10 Gold competent cell from -80 ℃ refrigerator; put on ice
  • Add 2 μl GFP generator DNA solution into competent cell
  • Place on ice, 30 min
  • Heat shock, 42 ℃, 90 sec
  • Chill on ice, 5 min
  • Add 800 μl LB medium (without antibiotics)
  • Incubate at 37 ℃, 1 hr
  • For TA ligation product, smear 100 μl 50 mg /ml Ampicilin, 100 μl 0.1 M IPTG and 50 μl X-gal over LB agar plate.For pSB1C3 ligation product, smear 100μl 34 mg /ml Chlorophenicol over the plate.
  • Bacteria solution centrifuge 3000 rpm, 5min
  • Remove 700 μl supernatant
  • Mix bacteria solution; Smear 100 μl over LB agar plate
  • Incubate at 37 ℃, overnight

Plasmid Extraction

  • Pick single colonies; each colony incubate with 3 ml LB medium (add antibiotics); 37℃, overnight
  • Centrifuge 7500 rpm, 1 min; remove supernatant
  • Resuspend bacteria with 200 μl PD1 buffer; resuspend bacterial pellet
  • Add 200 μl PD2 buffer; gently mix
  • Add 300 μl PD3 buffer; gently mix
  • Centrifuge 13000 rpm, 15 min
  • Transfer supernatant to kit filter
  • Add 400 μl W1 buffer; Centrifuge 13000 rpm, 1 min; Discard the flow-through
  • Add 600 μl Wash buffer; Centrifuge 13000 rpm, 1 min; Discard the flow-through
  • Centrifuge 13000 rpm, 5 min; air- dry
  • Dissolve DNA with 50 μl ddH2O; Centrifuge 13000 rpm,1 min
  • Storage at – 20℃

Double Enzyme Digestion

  • 16 μl ddH2O
  • 0.5 μl Enzyme 1
  • 0.5 μl Enzyme 2
  • 3 μl NEB 10X buffer
  • 0.3 μl NEB 10X BSA
  • 10 μl DNA
  • Incubate at 37 ℃, 3 hr