IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/06/16
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J45004-1: 59.1 ng/μL J45004-2: 72.9 ng/μL J45004-3: 88.1 ng/μL J45700-1: 146.7 ng/μL J45700-2: 187.4 ng/μL J45700-3: 175.5 ng/μL
Digestion with Enzymes
Team Genetic Fence
Performed miniprep of overnight cell cultures of 3 colonies each of wildtype LacI transformed bacteria and LacI+rapid degradation tail bacteria.
Minipreps performed according to pages 22-23 of the QIAprep Miniprep Handbook.
Blanked nanodrop sensor with 2μL EB buffer, then measured each of the 6 minipreped LacI plasmids.
LacI Plasmid Digest
We digested 15μL of each of the 6 samples with 1μLH2O, 1μL EcoR1, and 1μL Pst1 (high efficiency restriction enzymes).
Ran an E-gel of the digest results.
VP16 Colony Inoculation
Prepared 3 10cc tubes each containing .5μL LB+amp and cells from one of the three colonies of the VP16 cell culture of the previous night (colonies from the second-tube plate, the cells in which were heatshocked in the correct order).
Tubes were set to shake at 37°C overnight.
Preparing Glycerol Stocks of LacI wt and LacI+LVA
Prepared 2 cryo tubes, with the following labeling:
Tube 1) contains BBa_C0012, from plate 1, well 2O
On top: '#1'
On side: '6-16-10 MP pSB1A2 LacI+LVA'
Tube 2) contains BBa_I732100, from plate 2, well 10E
On top: '#2'
On side: '6-16-10 MP pSB1A3 LacI Wildtype'
Each cryo tube contains .5mL of the respective overnight cell culture (O2 colony #1 and E10 colony #1), and .mL of 80% glycerol.
Both Cryo tubes were placed in the box labeled 'Genetic Fence iGEM 2010' in the -80°C freezer.