Team:Stockholm/18 October 2010

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{{Stockholm/Top2}}
{{Stockholm/Top2}}
==Andreas==
==Andreas==
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 +
===Transfer of ProtA⋅His to pEX===
 +
 +
====Digestion====
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
| 
 +
!width="50"|Sample
 +
|-
 +
|10X FastDigest buffer
 +
|align="center"|1
 +
|-
 +
|Plasmid DNA
 +
|align="center"|7
 +
|-
 +
|dH<sub>2</sub>O
 +
|align="center"|0
 +
|-
 +
|FD XbaI
 +
|align="center"|1
 +
|-
 +
|FD PstI
 +
|align="center"|1
 +
|-
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|&nbsp;
 +
!10 &mu;l
 +
|}
 +
*Incubation: 37 &deg;C, 0:30
 +
*Inactivation: 80 &deg;C, 3:00
 +
 +
De-phosphorylated pre-digested/extracted pEX vector:
 +
*8 &mu;l extracted and digested (X+P) pEX vector DNA
 +
*1 &mu;l FD buffer
 +
*1 &mu;l FastAP (alkaline phosphatase)
 +
 +
*Incubation: 37 &deg;C, 0:30
 +
*Inactivation: 80 &deg;C, 3:00
 +
 +
====Ligation====
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
|10X T4 Ligase buffer
 +
|align="center"|2
 +
|-
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|Vector DNA
 +
|align="center"|1
 +
|-
 +
|Insert DNA
 +
|align="center"|11
 +
|-
 +
|dH<sub>2</sub>O
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|align="center"|5
 +
|-
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|T4 DNA ligase
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|align="center"|1
 +
|-
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|&nbsp;
 +
!20 &mu;l
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|}
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*Incubation: 22 &deg;C, 1 h
 +
 +
====Transformation====
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''Including three more transformations''
 +
 +
Modified quick-transformation protocol:
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*30 min on ice
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*50 &mu;l BL21
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*#2 &mu;l pEX.ProtA&sdot;his ligation mix
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*#0.5 &mu;l pEX.IgGp
 +
*#0.5 &mu;l pEX.SOD.RyC
 +
*#0.5 &mu;l pEX.hS.RyC
 +
 +
===PCR verification for Uppsala team===
 +
Helping the Uppsala team with a PCR verification of one of their assemblies.
 +
 +
Summed up their total construct length in pSB1x3 to 6566 bp.
 +
 +
*K1: C2 & C4 (x2)
 +
*K2: C2 & C5 (x2)
 +
 +
Standard colony PCR settings:
 +
*Elongation time: 10 min
 +
*Annealing temp: 55 &deg;C and 60 &deg;C
 +
 +
PCR run ON.

Revision as of 18:20, 18 October 2010

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Contents

Andreas

Transfer of ProtA⋅His to pEX

Digestion

  Sample
10X FastDigest buffer 1
Plasmid DNA 7
dH2O 0
FD XbaI 1
FD PstI 1
  10 μl
  • Incubation: 37 °C, 0:30
  • Inactivation: 80 °C, 3:00

De-phosphorylated pre-digested/extracted pEX vector:

  • 8 μl extracted and digested (X+P) pEX vector DNA
  • 1 μl FD buffer
  • 1 μl FastAP (alkaline phosphatase)
  • Incubation: 37 °C, 0:30
  • Inactivation: 80 °C, 3:00

Ligation

10X T4 Ligase buffer 2
Vector DNA 1
Insert DNA 11
dH2O 5
T4 DNA ligase 1
  20 μl
  • Incubation: 22 °C, 1 h

Transformation

Including three more transformations

Modified quick-transformation protocol:

  • 30 min on ice
  • 50 μl BL21
    1. 2 μl pEX.ProtA⋅his ligation mix
    2. 0.5 μl pEX.IgGp
    3. 0.5 μl pEX.SOD.RyC
    4. 0.5 μl pEX.hS.RyC

PCR verification for Uppsala team

Helping the Uppsala team with a PCR verification of one of their assemblies.

Summed up their total construct length in pSB1x3 to 6566 bp.

  • K1: C2 & C4 (x2)
  • K2: C2 & C5 (x2)

Standard colony PCR settings:

  • Elongation time: 10 min
  • Annealing temp: 55 °C and 60 °C

PCR run ON.

Retrieved from "http://2010.igem.org/Team:Stockholm/18_October_2010"