Team:Newcastle/13 July 2010

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==Conclusion==
==Conclusion==
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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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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 the feint second band we are unsure as to whether plasmid has fully digested, to that end we will leave the digest running overnight and run the samples on a gel tomorrow.  
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Revision as of 11:02, 10 August 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

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 in the way we desired (i.e. in front of RFP). 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 the feint second band we are unsure as to whether plasmid has fully digested, to that end we will leave the digest running overnight and run the samples on a gel tomorrow.

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