Team:Newcastle/13 July 2010

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Contents

LacI BioBrick Construction

Aims

To use PCR to extract lacI (promoter, ribosome-binding site (RBS) & coding sequence (CDS)) from plasmid pMutin4 and ligate into vector pSB1AT3 in front of red fluorescent protein (RFP).

Materials

Seven overnight cultures Restriction enzyme ecoR1

Protocol

  • E. coli DH5alpha was transformed again with our ligation mix.
  • The seven overnight cultures underwent plasmid extraction.
  • The seven plasmid extracts were then restriction digested with ecoR1 to linearise.
  • The seven digests were then run on a gel.

Results

Newcastle LacI Gel 3.PNG

This procedure was followed in order to determine whether our lacI had been ligated into the plasmid vector. The Gel shows bands in samples 3-7 that correspond to the size of pSB1AT3 plus the original RFP insert (4.5 Kbp) and in samples 1 and 2 that correspond to vector only (3.5 Kbp).

Conclusion

The conclusion that can be drawn from this gel is that the lacI has not been inserted into the plasmid as we desired. However due to some confusion as to the actual size of the vector a set of double digests will be performed tomorrow to try to extract insert form the plasmid if any is present.

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