Team:Brown/Notebook/October6

From 2010.igem.org

Wednesday, October 6, 2010

Transforming LacI ligation product into XL1B

Entire ligation products (50ul each trial, 2 trials) were added to competent cells and tranformation protocol was followed. After heatshock, LB was added and the cells were incubated at 37C for 1hr before plating as a recovery step to increase the chance of successful colonization. Final product (LacI in pSB1C3) was grown on chlor plates 50ul per plate.

Ran Gel of Colony PCR samples

A4, A5, C1, C4, C5, M4, M5 Only CI visible, and gel was "runny" --> Will need to redo PCR, ligation, selection.

To test PLac/Mnt hybrid promoter: 1) Digested pLac/Mnt with E+S 2) Digested J06702 with E+X 3) Digested TerTer (B0015) with E+X 4) Digested pTetR+RBS+LacI with E+S

1) 15μl pLac/Mnt to 500 ng

  • 0.5 μl BSA (100X)
  • 5 μl Buffer II
  • 2.5 μl EcoRI
  • 2.5 μl SpeI
  • 24.5 dH2O

2) 15μl J06702 to ~500ng

  • 0.5 μl BSA (100x)
  • 5 μl Buffer II
  • 2.5 μl EcoRI
  • 2.5 μl XbaI
  • 24.5 μl dH2O

3) 10 μl TerTer to ~600 ng

  • 0.5 μl BSA (100x)
  • 5 μl Buffer II
  • 2.5 μl EcoRI
  • 2.5 μl XbaI
  • 24.5 μl dH2O

4) 4 μl LacI + J13002 to 600 ng

  • 42.5 μl master mix
  • 2 μl EcoRI
  • 2 μl SpeI

Put in incubator at 37°C at 7:20 PM Removed at 8:50 PM Qiagen PCR cleanup Ligation of pLac(Mnt (insert) to J06702 (vector)) and pTetR+Rbs+LacI(insert) to terter(B0015)(vector) using 30 μl total volume protocol. 4°C at 10:33 PM.

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