Team:Brown/Notebook/August10

From 2010.igem.org

Tuesday, August 10, 2010

Miniprep of LovtapTwo WT overnight cultures

7 tubes run in parallel. Nanodrop concentrations:

  1. 249.2ng/ul
  2. 208.1ng/ul
  3. 192.5ng/ul
  4. 280.1ng/ul
  5. 261.3ng/ul
  6. 250.2ng/ul
  7. 211.6ng/ul

Digest of Lovtwo-Bgl with EcoRI

Ran a 1% agarose gel. Notice the colllapsed wells (did not significantly affect results).

Gel 81010.jpg

Interpretation - ligation failed. Will re-ligate and re-transform using Blue-white selection.

Re-ligation of LovTwo into pGEM

1:3 vector:insert ratio. 15ul total volume:

  • 7.5ul ligase buffer (2x)
  • 1ul T4 ligase
  • 0.5ul vector DNA
  • 1.5ul insert DNA
  • 4.5ul dH2O

Transformation of BL21 with LacI reporter construct (J04421)

We used BL21 because CFP expression will be more visible in a high protein expression strain. We used 800pg of DNA (0.5ul of 1.6ng/ul miniprep dilution).

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