E. coli Lab

Instructor: Rachit Jain

February 2013

-Training on PCR and heat shock transformation

March 2013

-PCR of GS gene with 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.