Revision as of 12:17, 10 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).

Single and Double digest of Plasmid extracts (single Xba1) Run digests on a gel Overnight culture of second round of transformants

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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'''. The experiments that were carried out [[Team:Newcastle/16_July_2010#Protocol|yesterday]] are being repeated today as there were extra and unexpected bands on the image of the gel.
+==Materials==
+*[[Team:Newcastle/14_July_2010|Second round of Transformants]]
+*[[Team:Newcastle/12_July_2010|Plasmid extracts]]
+*Restriction enzymes ''Xba''1 aand ''Pst''1
 ==Protocol==
-#Miniprep - the [[Team:Newcastle/Qiagen_Minipreps|miniprep]] protocol was followed.
+Single and Double digest of Plasmid extracts (single ''Xba''1)
-#Restriction digests - the [[Team:Newcastle/Restriction_digests|restriction digests]] protocol was followed to allow the length of the DNA to be checked using single and double digest.
+Run digests on a gel
-#*Single digest with Pst1
+Overnight culture of second round of transformants
-#*double digests with Pst1 and Xba1
-#Gel electrophoresis - the [[Team:Newcastle/Gel_electrophoresis|gel electrophoresis]] protocol was followed.
-#Set up liquid broth culture in 5 ml of LB - 1 colony from colonies 1-7. The protocol for [[Team:Newcastle/Growing_an_overnight_cultures| growing an overnight culture]] was followed.
+==Results==
 {{Team:Newcastle/footer}}