Revision as of 14:20, 6 August 2010

LacI BioBrick Construction

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).
The aim of today's experiment is to screen for the lacI insert. This could be done using PCR but that would require specific primers and is not as reliable.

Miniprep - the miniprep protocol was followed.
Restriction digests - the restriction digest protocol was followed.
- Single digest with Pst1
- double digests with Pst1 and Xba1
Gel electrophoresis

Set up liquid broth culture in 5 ml of LB - 1 colony from colonies 1-7

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 ==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).
+*The aim of today's experiment is to '''screen for the lacI insert'''. This could be done using PCR but that would require specific primers and is not as reliable.
-Screening for lacI insert:
 #Miniprep - the [[Team:Newcastle/Qiagen_Minipreps|miniprep]] protocol was followed.
 #Restriction digests - the [[Team:Newcastle/Restriction_digests| restriction digest]] protocol was followed.