Team:DTU-Denmark/Notebook/15 August 2013

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15 August 2013

Contents

lab 208


Main purpose


  • Plasmid isolation of CycAX, TAT3-2, TAT3-1a, Sec2, HAO, pZA21::RFP and pZA21::araBAD::RFP.

Who was in the lab


Ariadni, Helen, Kristian, Julia

Procedure


Plasmid isolation

Plasmid have been isolated following the protocol provided by ORIGENE PowerPrep HP Plasmid Miniprep Kit.

Glycerol stock cultures

-80C stock solution of Sec2, TAT3-1a, TAT3-2

DpnI treatment

DpnI treated the USER fragments for the SPl project since there were many colonies on the negative controls which can only arise from template DNA used in the PCR. This is removed by DpnI.

User ligation and transformation

Repeated ligation and transformation from yesterday with same reaction mix to get seperated colonies useable for the SPL project. Plating in a gradient: 5uL, 50 uL, 100uL

SPL project

Picked single colonies of araBAD SPL transformants and replated them to prepare for Biolector experiment to measure the strength of the expression.

Results


gel

  • 1 kb ladder
  • purification of the extraction fragment of Nir2
  • Nir2 USER touchdown, sample 1
  • Nir2 USER touchdown, sample 2
  • Nir2 USER touchdown, sample 3
  • Nir2 USER touchdown, sample 4
  • Nir2 USER touchdown, sample 5
  • Nir2 USER touchdown negative
  • 1 kb ladder

2013-08-15 nir2 user.jpg

The purification has the wrong length for Nir2 and was therefore discarded.

transformation

Yesterday's transformation yielded too many colonies that were inseparable, it will therefore be repeated and less volume will be plated.

Conclusion


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