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Team:Newcastle/14 July 2010
From 2010.igem.org
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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). | ||
| + | *To see whether the LacI insert is in the pSB1AT3 plasmid (same as yesterday). | ||
| + | ==Materials and Protocol== | ||
*Performed a gel electrophoresis with remaining PCR products + Controls with same restriction digests. | *Performed a gel electrophoresis with remaining PCR products + Controls with same restriction digests. | ||
| - | + | ====Transformation==== | |
| + | *''E. coli'' DH5alpha was transformed again with our plasmid. The [https://2010.igem.org/TeamNewcastleTransformation_of_E._coli#Protocol| transformation protocol] was followed. | ||
| - | + | ====Miniprep==== | |
| + | *For this step the [[Team:Newcastle/Qiagen Minipreps| Qiagen Minipreps]] protocol was followed. The 7 colonies that grew on the plates from yesterday were cultured ovenight on a shaking shelf at 37°C in universal tubes. | ||
| - | we followed Wendy's cards/Phil's lab-book | + | ====Restriction digests==== |
| + | *The [https://2010.igem.org/TeamNewcastleRestriction_digests| restriction digest] protocol was followed for this step. | ||
| + | |||
| + | (we followed Wendy's cards/Phil's lab-book) | ||
{{Team:Newcastle/footer}} | {{Team:Newcastle/footer}} | ||
Revision as of 06:36, 9 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).
- To see whether the LacI insert is in the pSB1AT3 plasmid (same as yesterday).
Materials and Protocol
- Performed a gel electrophoresis with remaining PCR products + Controls with same restriction digests.
Transformation
- E. coli DH5alpha was transformed again with our plasmid. The transformation protocol was followed.
Miniprep
- For this step the Qiagen Minipreps protocol was followed. The 7 colonies that grew on the plates from yesterday were cultured ovenight on a shaking shelf at 37°C in universal tubes.
Restriction digests
- The restriction digest protocol was followed for this step.
(we followed Wendy's cards/Phil's lab-book)
   
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