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Team:UGA-Georgia/NotebookE.coli
From 2013.igem.org
E. coli Lab
Instructor: Rachit Jain
February 2013
-Training on PCR and heat shock transformation
March 2013
-PCR of GS gene and histidine tag (histag).
-Cloning of GS-histag gene into pAW-50.
-Transformation of pAW-50 GS-histag into species XL1-Blue. Transformed species spread onto ampicillin plates
-Verification of GS-histag transformants via digestion and gel verification
April 2013
-Colonies for GS, AT, and GS+AT were selected from Methanococcus and corresponding genes were extracted. PCR and gel verification of GS, AT, and GS+AT genes. Results concluded successful in locating vectors from Methanococcus.
-PCR and gel verification of mCherry gene
-Cloning of mCherry gene into pAW-50 vector
-Verification of cloning via digestion and gel verification. After positive verification, ligation of digested products and heat shock transformation into XL1-Blue. XL1-Blue transformants spread onto ampicillin plates.
-XL1-Blue colonies picked and screened. Purification of pAW-50 mCherry from colonies, and stored in -20°C freezer.
June 2013
-Transformation of pAW-50 mCherry into XL1-Blue and creation of stock
-PCR of two mCherry sequences: one without RBS sequence (V2) and one containing RBS sequence (V4). Gel Verification of tools 1 and 2.
-Digestion and cloning of V2 and V4 into pAW-50 mCherry vector
July 2013
-Purification of digested vector, ligation, and transformation of vector into XL1-Blue. Transformants plated onto ampicillin plates.
-V2 and V4 colonies extracted and screened. Permanent stocks of V2B and V4B stored in -80°C freezer.
