Jessica Karen Wong/Notebook/2007-8-8

• Minipreped 2 samples of I2055-SX-B0030 for seq
  • Sent both samples (1 was fluor and 1 wasn't) for seq w/ VF & VR
• PCR cleaned I2056 digests (E/X, S/X, both N)
• Ligated I2056-ENX closed, I2056-SNX closed, I2056-EX-B34, I20550EX-B34
• Ligated in the presence of Xba and Spe restriction enzymes: I2055-SX-B32, I2056-SX-B32, I2056-SX-B34, I2055-SX-B34
  • Transformed w/ top10
  • Ran out of B0032 cut E/S so made an overnight of B0032 to later cut and ligate
• Digested E0240 S/N to go into F2620, prep I2055 S/X, prep I2055 E/X

PCR Digest Gel

• Ran a gel of the PCR product of each construct next to the Digest of that PCR to make sure digests were cutting in the right place
  • Loaded: lad sp I2057-EX, EX Digest, I2057-ES, ES digest, I2056-EX, EX digest, I2056-SX, SX digest, I2055-EX, I2055 EX digest, sp, I2055 SX digest
  • Didn't have any more PCR product of I2055-SX

Seq

• I2055-EX seems to have a 1bp mutation at 892 - bad
• I2055-SX has a few bp differences, breaks down towards 806 even w/ VR - bad
• I2057-EX-J116 is actually J102, I2057 part is fine after bp 13
  • I2057 first 12 bp's have strange double peaks in chromatogram, large peaks are wrong, small are right
• I2056-7, I2057-ENS, I2057-ENX all failed again

Made overnights of I2055-EX-B0032, I2056 colony 7, I2057-ENS, I2057-ENX, I2055-ENX-2, I2055-SNX-6, I2055-SNX-7 to sequence