User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/24

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Check overnight growth Good! I checked overnight growth of nine 10ml bottles with kanamycin and selected colonies. 4 bottles based on pUA66katE#A1#2, a verified colony that contains the katE insert 3 bottles from separate plates with colonies containing SAP treated vectors that have been ligased 2 bottle with a standard ligase My question was - whether the SAP treatment ensured the presence of my insert given that the colonies grew? I proceeded using 2 bottles of pUA66katE#A1#2 for miniprep, I kept one to preform assay and the last bottle was centrifuged and the cell pellets frozen. For the SAP treated vectors, 2 were used for miniprep. For the standard ligation 1 bottle was used for miniprep. All remaining bottles were centrifuged and the cell pellets frozen at -20ºC. Glycerol stock I decided to do a miniprep on 4 of 8 bottles: pAU66katE#A1#2-1 (known to contain the insert) pAU66katE#A1#2-2 (known to contain the insert) pUA66katE#A1, SAP pUA66katE#B1, SAP pUA66katE#2, Normal Ligase In order to preserve any sample that screened positive for the insert, I prepared glycerol stock. For each of the 5 samples: 700µl bacteria sample 300µl of 50% Glycerol Place in -80ºC freezer Miniprep Digestion