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From 2010.igem.org

Contents 1 Protocol 1.1 PCR (total volume: 50 μl ) 1.2 Gel Extraction- use Gel /PCR DNA Fragments Extraction Kit (Geneaid) 1.3 TA Cloning- use pGEM®-T Easy Vector (Promega) 1.4 Ligation on pSB1C3- use Elite T4 DNA ligase (Geneaid) 1.5 Transformation 1.6 Plasmid Extraction 1.7 Double Enzyme Digestion

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