Team:Newcastle/13 July 2010

From 2010.igem.org

Revision as of 10:26, 10 August 2010 by PhilipHall (Talk | contribs)

iGEM Homepage Newcastle University BacillaFilla Homepage Image Map

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


Protocol


Results

Newcastle LacI Gel 3.PNG

Figure 1: Gel electrophoresis of the seven plasmid extracts which have been digested with EcoR1.

  • Lane 1: 1 Kbp Molecular weight marker
  • Lane 2-8: Colonies 1-7 in order
  • Lane 9: 1 Kbp Molecular weight marker


This procedure was followed in order to determine whether our lacI had been ligated into the pSB1AT3. The gel shows bands for colonies 1 and 2 that indicate that they contain vector only (size 3 Kbp) and for 3-7 that indicate that they contain vector plus RFP (size 4.5 Kbp), as with the original plasmid stock.

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.

Newcastle University logo.png    Newcastle cbcb logo.pngNewcastle Biomedicine logo.gif    Team Newcastle CEG logo.gif
Newcastle iww logo.jpg  UNIPV Pavia Logo.gif  Newcastle BBSRC.gif    Newcastle Genevision logo.png Newcastle WelcomeTrust.jpg
FaceBook Icon