Jessica Karen Wong/Notebook/2007-7-3

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To Do

  • Colony PCR I2056 Tet
  • Replate I2056
  • Order new primer for I2055
  • Make overnights of other I2055 colonies for sequencing
  • Run gel of I2056 and T9002

I2055

  • Took out gradient PCR
  • Checked sequencing using AlignX and Finch TV on Jason's computer
  • I2055 had not ligated the R0040 to the I3401, aka the promoter was missing
  • We went back to the gel image to see if any other colony pcr's look a bit bigger and may include R0040
  • Made overnights of colonies 3,4, and 6 to miniprep for sequencing tomorrow
  • 5ml overnights were made with 5ul Amp and 5ul Kan b/c is on a 1AK3 plasmid
  • Also designed primers to PCR R0040 in if none of the colonies were successfully ligated
    • Only ordered a forward primer with Tail including Mfe1 and R0040
    • format: 8-mer.MfeI.promoter+mix.part of gfp (Tm)
    • CTTAGTAG.CAATTG.tccctatcagtgatagagattgacatccctatcagtgatagagatactgagcactactag.ATGCGTAAAGGAGAAGAACTTTTC (52.4)

I2056

  • Saw a colony on the Tet plate
  • Did a 10ul colony PCR on it, but result seemed slightly evaporated
  • Redoing the colony PCR overnight
  • Spun down and replated the rest of the transformants on both Tet and Chlor and are growing overnight

Gel

  • Gel from 7/3/07
  • Ran a gel of the I2056 colony PCR and 10 ul of the T9002 100ul preparatory PCR
  • Loaded gel L to R: Ladder __ T9002 I2056 Ladder
  • Did a post stain with cybr gold
  • Scarred T9002 looks the right size
  • Redoing I2056



2 log ladder