Team:Brown/Notebook/September23

From 2010.igem.org

Thursday, September 23, 2010

Miniprep of LacI J13002 out of XL1B overnight culture

Eluted in 35μl buffer EB. Note: Lyseblue not added to Buffer P1, but it should be fine.

15 overnight cultures in Amp at 230 PM

  • AraC1, AraC2, AraC3, AraC4, AracC5
  • Mnt1, Mnt2, Mnt3, Mnt4, Mnt5
  • CI1, CI2, CI3, CI4, CI5

(All of the above ligated into pGEM)

Corresponding are 15 PCR tubes with bottoms scratches with colonies A1, A2, A3... Will do colony PCR with this when primers are recovered (stored now in 4°C).

Transformation

Transformation of ligation products from 9/22: Tat-L into pGEM and Tat-L into pSB1A3

  • Added 10 μl of ligation product to XL1 cells
  • Used blue-white selection for Tat-L in pGEM
  • incubated at 37°C, start time at 4:50 PM.

Plate Observations

Random screening of plate from 9/18 containing pBlueScript+inserted Tat-L

  • All colonies present show blue, implying unsuccessful insertion.
  • However, small size of Tat-L insert suggests possibly that blue-white selection will not be disrupted.
  • Will pick 8 random colonies, PCR with Tat-L primer set. If colony contains Tat-L sequence, means amplified DNA will show on gel.
  • At same time, grow overnights with all 8 potential colonies.
  • Placed 8 PCR reactions in thermocycler (lost sample #5).
  • Overnighted 8 colonies (lost #5) in 37°C. Start time: 5:10 PM
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