Jessica Karen Wong/Notebook/2007-7-3

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