Jessica Karen Wong/Notebook/2007-7-8

Colony PCRs

• 

  Gel from 7/8/07

• Ran a gel of the overnight Colony PCR's
• L to R: Ladder; T9002-3K3 (4,3,2,1); I2055-1AK3 (1,2,3,4); E0240-3K3 (4,3,2,1); E0240-1AK3 (1,2); Ladder
• T9002 was to check if it is scarred, I2055 was to see if it is constructed (religation, not pcr-ing promoter), both E0240's scarred
• T9002 should be over 2kb but shows as only 400bp
• I2055 looks the right size (except for #2 which I probably messed up)
• E0240-1AK3 looks fine but 3K3 is also about 500bp
• Most likely 3K3 is the problem
• Made overnights of each type to prep for sequencing tomorrow
• Also overnighted 3K3 plasmid from original registry plate

I2055 w/ PCRed in Promoter

• Heat Shocked overnight double digest with Mfe1 and Nsi1
• Ligated with 1AK3
• Transformed 3ul of ligation and plated on Kan
• Started to ligate with 3K3 but since 3K3 is acting up, was discarded

I2056

• Overnights in LB and Tet worked
• Strange, must have somehow lost Amp resistance
• Made a glycerol
• Minipreped w/ 8.5 water for sequencing and w/ EB