Team:TzuChiU Formosa/Notebook

From 2010.igem.org

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This is a template page. READ THESE INSTRUCTIONS.
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You are provided with this team page template with which to start the iGEM season.  You may choose to personalize it to fit your team but keep the same "look." Or you may choose to take your team wiki to a different level and design your own wiki.  You can find some examples <a href="https://2008.igem.org/Help:Template/Examples">HERE</a>.
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You <strong>MUST</strong> have a team description page, a project abstract, a complete project description, a lab notebook, and a safety page.  PLEASE keep all of your pages within your teams namespace. 
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==Protocol==
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===PCR (total volume: 50 μl )===
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:* 25 μl Green master mix (Promega)
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:* 2 μl each primer
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:* 2 μl template
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:* 19 μl ddH2O
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:* Cycle 95/2 min; 30x(95/1 min;56/1 min;72/2 min); 72/7 min
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{|align="justify"
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===Gel Extraction- use Gel /PCR DNA Fragments Extraction Kit (Geneaid)===
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|You can write a background of your team here.  Give us a background of your team, the members, etc.  Or tell us more about something of your choosing.
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:* Loading the PCR products into agarose gel; run gel with 1X TAE buffer
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|[[Image:TzuChiU_Formosa_logo.png|200px|right|frame]]
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:* Excise the agarose gel slice containing relevant DNA fregments
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|-
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:* Transfer gel slice to a 1.5 ml microcentrifuge tube
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|
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:* Add 500 μl DF buffer; incubate at 56 ℃ until gel slice has been completely dissolved
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''Tell us more about your project.  Give us background.  Use this is the abstract of your project.  Be descriptive but concise (1-2 paragraphs)''
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:* Transfer 800 μl gel mixture to DF column
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|[[Image:TzuChiU_Formosa_team.png|right|frame|Your team picture]]
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:* Centrifuge 13000 rpm, 1 min
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|-
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:* Discard the flow-through and place the DF column back
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|
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:* Add 400 μl W1 buffer; Centrifuge 13000 rpm, 1 min; Discard the flow-through
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|align="center"|[[Team:TzuChiU_Formosa | Team Example]]
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:* Add 600 μl wash buffer; Centrifuge 13000 rpm, 1 min; Discard the flow-through
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|}
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:* Centrifuge 13000 rpm, 5 min; air- dry
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:* Add 50 μl ddH2O; Centrifuge 13000 rpm,1 min
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:* Storage at – 20℃
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<!--- The Mission, Experiments --->
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===TA Cloning- use pGEM®-T Easy Vector (Promega)===
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:* 1μl pGEM®-T Easy Vector
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:* 5μl 2X rapid ligation buffer
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:* 1μl T4 DNA ligase
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:* 3μl insert
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:* 4 ℃, overnight
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{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"
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===Ligation on pSB1C3- use Elite T4 DNA ligase (Geneaid)===
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!align="center"|[[Team:TzuChiU_Formosa|Home]]
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:* 9 μl insert
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!align="center"|[[Team:TzuChiU_Formosa/Team|Team]]
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:* 3 μl vector
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!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=TzuChiU_Formosa Official Team Profile]
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:* 2 μl 10X ligation buffer A
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!align="center"|[[Team:TzuChiU_Formosa/Project|Project]]
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:* 2 μl 10X ligation buffer B
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!align="center"|[[Team:TzuChiU_Formosa/Parts|Parts Submitted to the Registry]]
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:* 1 μl Elite T4 DNA ligase
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!align="center"|[[Team:TzuChiU_Formosa/Modeling|Modeling]]
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:* 3 μl ddH2O
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!align="center"|[[Team:TzuChiU_Formosa/Notebook|Notebook]]
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:* 16℃, overnight
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!align="center"|[[Team:TzuChiU_Formosa/Meeting Minutes|Meeting Minutes]]
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!align="center"|[[Team:TzuChiU_Formosa/Safety|Safety]]
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|}
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===Transformation===
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:* Take out XL-10 Gold competent cell from -80 ℃ refrigerator; put on ice
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:* Add 2 μl GFP generator DNA solution into competent cell
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:* Place on ice, 30 min
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:* Heat shock, 42 ℃, 90 sec
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:* Chill on ice, 5 min
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:* Add 800 μl LB medium (without antibiotics)
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:* Incubate at 37 ℃, 1 hr
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:* 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.
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:* Bacteria solution centrifuge 3000 rpm, 5min
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:* Remove 700 μl supernatant
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:* Mix bacteria solution; Smear 100 μl over LB agar plate
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:* Incubate at 37 ℃, overnight
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==Notebook==
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===Plasmid Extraction===
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:* Pick single colonies; each colony incubate with 3 ml LB medium (add antibiotics); 37℃, overnight
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:* Centrifuge 7500 rpm, 1 min; remove supernatant
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:* Resuspend bacteria with 200 μl PD1 buffer; resuspend bacterial pellet
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:* Add 200 μl PD2 buffer; gently mix
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:* Add 300 μl PD3 buffer; gently mix
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:* 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
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:* Storage at – 20℃
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You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer. It is a very useful organizational tool as well.
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===Double Enzyme Digestion===
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:* 16 μl ddH2O
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:* 0.5 μl Enzyme 1
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:* 0.5 μl Enzyme 2
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:* 3 μl NEB 10X buffer
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:* 0.3 μl NEB 10X BSA
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:* 10 μl DNA
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:* Incubate at 37 ℃, 3 hr

Latest revision as of 07:11, 4 December 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
Retrieved from "http://2010.igem.org/Team:TzuChiU_Formosa/Notebook"