Team:Stockholm/18 October 2010

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Revision as of 18:20, 18 October 2010 by AndreasConstantinou (Talk | contribs)


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.