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Monday, August 2, 2010

(Re?)digestion of RBS34

50ul total volume

• 5ul NEB 10x buffer 2
• 39.5ul dH2O
• 1ul EcoRI
• 1ul SpeI
• 0.5ul 100x BSA
• 3ul miniprep of RBS34

Gel appears to be cloudy and contaminated

Kan/Tet plate test results

None of the hand-contamination plates grew. XL1s on tet plates grew, but not BL21s. From this, we conclude that the XL1Bs may be contaminated with Tet resistance.

PCR of reporter system

Rehydration of primers

• Fwd - 156ul dH2O added
• Rev - 171.2ul dH2O added

PCR

50ul total volume

• 2ul fwd primer (10uM)
• 2ul reverse primer (10uM)
• 1ul template DNA
• 25ul master mix
• 20ul dH2O

Transformation of reporter constructs

• LacI reporter - from iGEM HQ, part J04421 miniprep product
• AraC reporter - from ligation on 7/29 of pAraC and J06702

3ul of each DNA in XL1Bs, standard 30min incubation and 1min heatshock.

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Transformation of AraC and bistable partin DH5alpha on Kan plates, 50ug/mL