Jessica Karen Wong/Notebook/2007-7-7

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  • Took out overnight transformation plates
    • both the sequential and double digests of T9002 and 3K3 had lots of colonies
    • E0240-3K3 had lots of colonies, E0240-1AK3 had two
    • I2055-1AK3 had lots of colonies, I-1AT3 and I-1AC3 had none

  • Made cell suspensions of 4 T-3K3 colonies, 4 I-1AK3 colonies, 4 E-3K3, and 2 E-1AK3
  • Did a 10 ul colony PCR on each cell suspensions
    • Used 2min elongation time because T9002 is 2261bp b/t VF and VR

  • Ran gel of I2055 and got a bright band just under 1kb
    • Not sure if it contains promoter or not
  • PCR cleaned I2055
  • Double digested I2055 with Mfe1 and Nsi1
    • Used buffer 2 and 4ul DNA

  • Overnight liquid cultures of I2056 still didn't grow (maybe lost Amp resistance?)
  • Made new overnights of I2056 containing only Tet

  • Did a 2-way ligation of T9002 and 1AK3
  • Transformed 3ul of the ligation and plated on Kan