Team:Newcastle/13 July 2010
From 2010.igem.org
(Difference between revisions)
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==Aims== | ==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). | + | *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). |
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*Seven [[Team:Newcastle/12_July_2010|overnight cultures]] | *Seven [[Team:Newcastle/12_July_2010|overnight cultures]] | ||
- | *Restriction enzyme | + | *Restriction enzyme ''Eco''R1 |
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*''E. coli'' DH5α was [[Team:Newcastle/Transformation_of_E._coli|transformed]] again with our ligation mix. | *''E. coli'' DH5α was [[Team:Newcastle/Transformation_of_E._coli|transformed]] again with our ligation mix. | ||
*The seven overnight cultures underwent [[Team:Newcastle/Qiagen Minipreps|plasmid extraction]]. | *The seven overnight cultures underwent [[Team:Newcastle/Qiagen Minipreps|plasmid extraction]]. | ||
- | *The seven plasmid extracts were then [https://2010.igem.org/TeamNewcastleRestriction_digests| restriction digested] with | + | *The seven plasmid extracts were then [https://2010.igem.org/TeamNewcastleRestriction_digests| restriction digested] with ''Eco''R1 to linearise. |
*The seven digests were then run on a gel. | *The seven digests were then run on a gel. | ||
Revision as of 09:58, 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
- Seven overnight cultures
- Restriction enzyme EcoR1
Protocol
- E. coli DH5α 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
This procedure was followed in order to determine whether our lacI had been ligated into the plasmid vector.
The gel shows bands in lanes:
- 2-3 (samples 1-2) that correspond to vector only (3.5 Kbp) and
- 4-8 (samples 3-7) that correspond to the size of pSB1AT3 plus the original RFP insert (4.5 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.