Jessica Karen Wong/Notebook/2007-8-6

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