Jessica Karen Wong/Notebook/2007-8-6

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Large Gel
  • Ran a gel of overnight pcr's
  • large gel:
    • Row 1 : E0240 M1 ES (4-1), E0240 M2 ES (1-4), SP LAD SP E0240-ES-E2 (4-1)E0240-EX-E2 (1-4)
    • Row 2 : F2620-3K3-1 (4-1), F2620-3K3-2 (1-4), SP LAD SP F2620-3K3-3 (4-1) E0240 M2-EX (1-4)
Small Gel
  • small gel: LAD SP E0240-E1-EX (4-1), E0240-ES-E1 (2-1), E0240-EX-M1 (2-1)
  • Overnighted F2620-3K3-2 colony 4, e0240-ES (M1 colony 1, M2 col 2, E2 col 1),E0240-EX (E2 col 2, E1 col 2)

  • Took out overnight liquid cultures and saw that I2057-EX-J116 was the only RFP culture that was red
  • Minipreped sealed I2055 EX, sealed I2055 SX, I2057-J116
  • Sent those 3 for sequencing with VF and VR
  • Made new I2057-EX-J100, I2057-EX-R0040, I2057-SX-R0040 from red colonies on each plate

  • Looked at the I2055-various RBS plates on the gel imager and saw very few fluorescent colonies
  • Realized that we forgot the restriction enzyme when ligating the RBS's in
  • Made an overnight of I2055-SX-B0031 which fluoresced
  • Streaked out a plate of I2055-SX-B0030 that had a small fluorescent area

  • Religated B0032-I2055-EX
  • Added .5ul of each S and X restriction enzyme to ligation mix of I2055-SX-B0032, 31, and 30
  • Transformed all 4 ligations into Top10 and plated

  • PCR cleaned B0034 digest
  • Was going to ligate into PCR of I2055-SX, but didn't have enough DNA
  • Digesting I2055-SX Spe/Xba
  • PCR cleaned B0034 digest

Got sequencing back

  • I2057-ES is perfect, complete coverage, total go ahead
  • I2057-EX rev has 1 N at 179, fwd failed and is being repeated
  • I2056-4,5,6 all have either an insertion or deletion at beginning - disregard
  • I2056-7 looks perfect until 864, has fwd scar, rev failed and is being repeated

Set up BB PCR of I2056-7 both EX and SX