Team:Stockholm/30 September 2010

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{{Stockholm/Top2}}
{{Stockholm/Top2}}
==Andreas==
==Andreas==
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 +
===Transfer of nCPP⋅SOD⋅His.RBS.yCCS operon to pEX===
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 +
====Digestions====
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#pSB1K3.nTAT⋅SOD⋅His.RBS.yCCS
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#*Clones 2 & 3
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#pSB1K3.nTra10⋅SOD⋅His.RBS.yCCS
 +
#*Clones 1 & 2
 +
#pSB1K3.nLMWP⋅SOD⋅His.RBS.yCCS
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#*Clones 2 & 3
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 +
 +
{|border="1" cellpadding="1" cellspacing="0"
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| 
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|align="center" width="50"|'''1''':2
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|align="center" width="50"|'''1''':3
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|align="center" width="50"|'''2''':1
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|align="center" width="50"|'''2''':2
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|align="center" width="50"|'''3''':2
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|align="center" width="50"|'''3''':3
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|-
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|10X FastDigest buffer
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|align="center"|2
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|align="center"|2
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|align="center"|2
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|align="center"|2
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|align="center"|2
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|align="center"|2
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|-
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|DNA (1 μg)
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|align="center"|5.2
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|align="center"|4.1
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|align="center"|2.5
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|align="center"|3.3
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|align="center"|4
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|align="center"|4.1
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|-
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|dH<sub>2</sub>O
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|align="center"|10.8
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|align="center"|11.9
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|align="center"|13.5
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|align="center"|12.7
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|align="center"|12
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|align="center"|11.9
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|-
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|FD XbaI
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|align="center"|1
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|align="center"|1
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|align="center"|1
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|align="center"|1
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|align="center"|1
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|align="center"|1
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|-
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|FD PstI
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|align="center"|1
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|align="center"|1
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|align="center"|1
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|align="center"|1
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|align="center"|1
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|align="center"|1
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|-
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|&nbsp;
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!20 &mu;l
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!20 &mu;l
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!20 &mu;l
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!20 &mu;l
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!20 &mu;l
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!20 &mu;l
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|}
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*Incubation: 37 &deg;C, 1.45
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*Inactivation: 80 &deg;C, 20 min
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====Ligations====
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*Vector: [Dig pEX.RFP X+P
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{|border="1" cellpadding="1" cellspacing="0"
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|&nbsp;
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|align="center" width="50"|pEX.'''1''':2
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|align="center" width="50"|pEX.'''1''':3
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|align="center" width="50"|pEX.'''2''':1
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|align="center" width="50"|pEX.'''2''':2
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|align="center" width="50"|pEX.'''3''':2
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|align="center" width="50"|pEX.'''3''':3
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|-
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|10X T4 Ligase buffer
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|align="center"|2
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|align="center"|2
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|align="center"|2
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|align="center"|2
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|align="center"|2
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|align="center"|2
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|-
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|Vector DNA
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|align="center"|1.5
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|align="center"|1.5
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|align="center"|1.5
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|align="center"|1.5
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|align="center"|1.5
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|align="center"|1.5
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|-
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|Insert DNA
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|align="center"|8
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|align="center"|8
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|align="center"|8
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|align="center"|8
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|align="center"|8
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|align="center"|8
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|-
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|dH<sub>2</sub>O
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|align="center"|7.5
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|align="center"|7.5
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|align="center"|7.5
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|align="center"|7.5
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|align="center"|7.5
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|align="center"|7.5
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|-
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|T4 DNA ligase
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|align="center"|1
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|align="center"|1
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|align="center"|1
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|align="center"|1
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|align="center"|1
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|align="center"|1
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|-
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|&nbsp;
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!20 &mu;l
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!20 &mu;l
 +
!20 &mu;l
 +
!20 &mu;l
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!20 &mu;l
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!20 &mu;l
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|}
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*Incubation: 22 &deg;C, 15 min
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====Transformation====
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Quick transformation
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*1 &mu;l ligation mix
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*50 &mu;l 0.1 M IPTG
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**pEX.1:2 (pEX.nTAT&sdot;SOD&sdot;His.RBS.yCCS 2)
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**pEX.1:3 (pEX.nTAT&sdot;SOD&sdot;His.RBS.yCCS 2)
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**pEX.2:1 (pEX.nTra10&sdot;SOD&sdot;His.RBS.yCCS 1)
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**pEX.2:2 (pEX.nTra10&sdot;SOD&sdot;His.RBS.yCCS 2)
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**pEX.3:2 (pEX.nLMWP&sdot;SOD&sdot;His.RBS.yCCS 2)
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**pEX.3:3 (pEX.nLMWP&sdot;SOD&sdot;His.RBS.yCCS 3)
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===Transformation of BL21===
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Quick transformation
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*50 &mu;l competent cells
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*0.5 &mu;l plasmid
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**pEX.nTra10&sdot;SOD&sdot;His
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**pEX.nLMWP&sdot;SOD&sdot;His

Revision as of 19:34, 30 September 2010

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Contents

Andreas

Transfer of nCPP⋅SOD⋅His.RBS.yCCS operon to pEX

Digestions

  1. pSB1K3.nTAT⋅SOD⋅His.RBS.yCCS
    • Clones 2 & 3
  2. pSB1K3.nTra10⋅SOD⋅His.RBS.yCCS
    • Clones 1 & 2
  3. pSB1K3.nLMWP⋅SOD⋅His.RBS.yCCS
    • Clones 2 & 3


  1:2 1:3 2:1 2:2 3:2 3:3
10X FastDigest buffer 2 2 2 2 2 2
DNA (1 μg) 5.2 4.1 2.5 3.3 4 4.1
dH2O 10.8 11.9 13.5 12.7 12 11.9
FD XbaI 1 1 1 1 1 1
FD PstI 1 1 1 1 1 1
  20 μl 20 μl 20 μl 20 μl 20 μl 20 μl
  • Incubation: 37 °C, 1.45
  • Inactivation: 80 °C, 20 min

Ligations

  • Vector: [Dig pEX.RFP X+P
  pEX.1:2 pEX.1:3 pEX.2:1 pEX.2:2 pEX.3:2 pEX.3:3
10X T4 Ligase buffer 2 2 2 2 2 2
Vector DNA 1.5 1.5 1.5 1.5 1.5 1.5
Insert DNA 8 8 8 8 8 8
dH2O 7.5 7.5 7.5 7.5 7.5 7.5
T4 DNA ligase 1 1 1 1 1 1
  20 μl 20 μl 20 μl 20 μl 20 μl 20 μl
  • Incubation: 22 °C, 15 min

Transformation

Quick transformation

  • 1 μl ligation mix
  • 50 μl 0.1 M IPTG
    • pEX.1:2 (pEX.nTAT⋅SOD⋅His.RBS.yCCS 2)
    • pEX.1:3 (pEX.nTAT⋅SOD⋅His.RBS.yCCS 2)
    • pEX.2:1 (pEX.nTra10⋅SOD⋅His.RBS.yCCS 1)
    • pEX.2:2 (pEX.nTra10⋅SOD⋅His.RBS.yCCS 2)
    • pEX.3:2 (pEX.nLMWP⋅SOD⋅His.RBS.yCCS 2)
    • pEX.3:3 (pEX.nLMWP⋅SOD⋅His.RBS.yCCS 3)

Transformation of BL21

Quick transformation

  • 50 μl competent cells
  • 0.5 μl plasmid
    • pEX.nTra10⋅SOD⋅His
    • pEX.nLMWP⋅SOD⋅His
Retrieved from "http://2010.igem.org/Team:Stockholm/30_September_2010"