iGEM 2010 — Team TzuChiU_Formosa

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# Team:TzuChiU Formosa/Notebook

### From 2010.igem.org

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| Contents   * [1 Protocol](#Protocol)   + [1.1 PCR (total volume: 50 μl )](#PCR_.28total_volume:_50_.CE.BCl_.29)   + [1.2 Gel Extraction- use Gel /PCR DNA Fragments Extraction Kit (Geneaid)](#Gel_Extraction-_use_Gel_.2FPCR_DNA_Fragments_Extraction_Kit_.28Geneaid.29)   + [1.3 TA Cloning- use pGEM®-T Easy Vector (Promega)](#TA_Cloning-_use_pGEM.C2.AE-T_Easy_Vector_.28Promega.29)   + [1.4 Ligation on pSB1C3- use Elite T4 DNA ligase (Geneaid)](#Ligation_on_pSB1C3-_use_Elite_T4_DNA_ligase_.28Geneaid.29)   + [1.5 Transformation](#Transformation)   + [1.6 Plasmid Extraction](#Plasmid_Extraction)   + [1.7 Double Enzyme Digestion](#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

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