IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/06/16

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Team Flavour

MiniPrep

  • MiniPrep of Wintergreen parts from Registry (J45004 and J45700) following Qiagen MiniPrep protocol. MiniPrep three samples of each part.
    • DNA concentrations:
         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

  • For J45004, we added: 9μL DNA, 1μL buffer, 1μL xbaI restriction enzyme (slow), and .5μL pstI restriction enzyme (fast).
  • For J45700, we added 5μL DNA, 4μL H2O, 1μL buffer, 1μL xbaI restriction enzyme (slow), and .5μL pstI restriction enzyme (fast).
  • We let mixtures sit for 30 minutes due to the use of xbaI restriction enzyme (slow). After the 30 minutes, we added 2.5μL dye to each mix.

Gel

  • We loaded 12.5μL of 1kb ladder to well 1 of the gel (numbered left to right). We then loaded 12.5μL of J45004-1, J45004-2, and J45004-3 to wells 2,3, and 4, respectively. We loaded J45700-1, J45700-2, J4500-3 in wells 5, 6, and 7, respectively (see images below for well locations).
  • Ran on 1% agarose gel.

BBa_J45700 BBa_J45700


Team Genetic Fence

LacI Miniprep

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.

LacI Nanodrop

Blanked nanodrop sensor with 2μL EB buffer, then measured each of the 6 minipreped LacI plasmids.

Plasmid Quantity (ng/μL) 260/280
O2 #1 77.1 1.81
O2 #2 90.1 1.9
O2 #3 50.4 1.95
E10 #1 99.6 1.93
E10 #2 104.6 1.92
E10 #3 58.6 1.98

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.

Lane # 1 2 3 4 5 6 7 8 9 10
O2 #1 ladder O2 #1 O2 #2 O2 #3 E10 #1 E10 #2 E10 #3

LacI digest Gel 2010-06-16 16hr 34min.jpg

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.