16/09/13

From 2013.igem.org

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(→Ligation of digested pSB1C3 backbone to Tod F, X and Tob G)
 
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==Digesting pSB1C3 backbone==
==Digesting pSB1C3 backbone==
*Digesting for ligation with the already digested Tod operon PCR products
*Digesting for ligation with the already digested Tod operon PCR products
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*Exluding the DpnI from the master mix and adjusting the volume of water accordingly
*Exluding the DpnI from the master mix and adjusting the volume of water accordingly
*Protocol can be found at: [http://parts.igem.org/Help:Protocols/Linearized_Plasmid_Backbones]
*Protocol can be found at: [http://parts.igem.org/Help:Protocols/Linearized_Plasmid_Backbones]
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==Ligation of digested pSB1C3 backbone to Tod F, X and Tob G==
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*Using Promega protocol to which changes were made
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{|border=1
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|Reagents||Tod F||Tod X||Tob G||Positive control||Background control
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|-
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|2x Rapid Ligation buffer||5ul||5ul||5ul||5ul||5ul
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|-
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|pSB1C3 digest (50ng)||2ul||2ul||2ul||2ul||2ul
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|-
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|PCR product||3.3ul||3ul||3.8ul||-||-
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|-
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|Control insert DNA||-||-||-||2ul||-
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|-
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|T4 DNA ligase||1ul||1ul||1ul||1ul||1ul
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|-
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|Deionized water for final volume of 10ul||-||-||-||-||1ul
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|}
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*Mix reactions by pipetting. Incubate the reactions for 1h at room temperature.
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*Control insert DNA from the transformation efficiency kit provided by iGEM
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==Transformation of TOD genes ligations with the digested pSB1C3 using the pGEM-T Vector System I-A3600 (changes were made to the protocol)==
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*Centrifuge the ligations reactions briefly.
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*Add 5ul of each ligation reaction to a sterile 1.5ml
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*Thaw the competent cells on ice. Mix the cells by gently flicking the tube.
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*Carefully transfer 50ul of the competent cells to the ligation reaction tubes.
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*Incubate the tubes on ice for 20 minutes
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*Heat-shock the cells for 45-50 seconds in a water bath at exactly 42C degrees. Do not Shake. Immediately return the tubes to ice for 2 minutes.
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*Add 900ul of room temperature SOC medium to the ligation reaction transformations. incubate for 1.5 hours at 37C with shaking.
==Digestion of the TOD genes PCR products==
==Digestion of the TOD genes PCR products==
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==Running the backbone and RFP on gel==
==Running the backbone and RFP on gel==
*on 1% gel to check if the gel extraction worked.
*on 1% gel to check if the gel extraction worked.
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*gel shows that extraction worked
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**2kb band representing the backbone is visible as well as 1kb representing the RFP
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==Digestion of the pGEM plasmid gel extracted on the [[13/09/13]]==
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The samples were digested with the enzymes EcoRI-HF and PstI-HF
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'''Master Mix for 8 reactions'''
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==Digestion of the pGEM plasmid extracted on the [[13/09/13]]==
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*16 ul of cutsmart buffer
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*4 ul of EcoRI
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*4 ul of PstI
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*88 ul of dH2O
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**14 ul of master mix was added to each reaction

Latest revision as of 08:49, 17 September 2013

Home Team Official Team Profile Project Parts Submitted to the Registry Modeling Notebook Safety Attributions


Contents

Digesting pSB1C3 backbone

  • Digesting for ligation with the already digested Tod operon PCR products
  • Following the iGEM official protocol
  • Using EcoRI-HF and PstI-HF
  • Exluding the DpnI from the master mix and adjusting the volume of water accordingly
  • Protocol can be found at: [http://parts.igem.org/Help:Protocols/Linearized_Plasmid_Backbones]

Ligation of digested pSB1C3 backbone to Tod F, X and Tob G

  • Using Promega protocol to which changes were made
ReagentsTod FTod XTob GPositive controlBackground control
2x Rapid Ligation buffer5ul5ul5ul5ul5ul
pSB1C3 digest (50ng)2ul2ul2ul2ul2ul
PCR product3.3ul3ul3.8ul--
Control insert DNA---2ul-
T4 DNA ligase1ul1ul1ul1ul1ul
Deionized water for final volume of 10ul----1ul
  • Mix reactions by pipetting. Incubate the reactions for 1h at room temperature.
  • Control insert DNA from the transformation efficiency kit provided by iGEM

Transformation of TOD genes ligations with the digested pSB1C3 using the pGEM-T Vector System I-A3600 (changes were made to the protocol)

  • Centrifuge the ligations reactions briefly.
  • Add 5ul of each ligation reaction to a sterile 1.5ml
  • Thaw the competent cells on ice. Mix the cells by gently flicking the tube.
  • Carefully transfer 50ul of the competent cells to the ligation reaction tubes.
  • Incubate the tubes on ice for 20 minutes
  • Heat-shock the cells for 45-50 seconds in a water bath at exactly 42C degrees. Do not Shake. Immediately return the tubes to ice for 2 minutes.
  • Add 900ul of room temperature SOC medium to the ligation reaction transformations. incubate for 1.5 hours at 37C with shaking.

Digestion of the TOD genes PCR products

The PCR products of TODX, TODF and TOBG were digested with the enzymes EcoRI-HF and PstI-HF.

Protocol

SampleDNA volume (ul)cutsmart buffer (ul)EcoRI (ul)PstI (ul)dH2O (ul)
TODX8.720.50.58.3
TODF8.320.50.58.7
TOBG820.50.59
  • The samples were incubated for 1 hour at 37C.
  • Heat kill the enzymes at 80C for 20 minutes.

Running the backbone and RFP on gel

  • on 1% gel to check if the gel extraction worked.
  • gel shows that extraction worked
    • 2kb band representing the backbone is visible as well as 1kb representing the RFP

Digestion of the pGEM plasmid gel extracted on the 13/09/13

The samples were digested with the enzymes EcoRI-HF and PstI-HF

Master Mix for 8 reactions

  • 16 ul of cutsmart buffer
  • 4 ul of EcoRI
  • 4 ul of PstI
  • 88 ul of dH2O
    • 14 ul of master mix was added to each reaction
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