Jessica Karen Wong/Notebook/2007-7-16

Gel

 * Ran a gel of the overnight colony PCR's of T9002-3K3
 * Loaded T9002-3K3 from 7-13 (1-8), space, ladder, space, same 9-16 in top lane
 * Loaded T9002-3K3-D from 7-11 (1-8), space ladder space t9002-3k3-s from 7-11 (1-8)
 * Got varying sizes but none large enough

T9002 Pieces

 * Got the primers to PCR Eco/Xba, Xba tails onto the pieces of T9002 aka F2620 and E0240
 * Diluted primers to 40 uM to PCR on the tails
 * Made 1 100ul rxn of each with vent using a 3x dilution of DNA
 * E0240 was run at 53.5 and F2620 at 53
 * Ran a gel of it, F2620 looks the right size, E0240 is too small
 * Redoing the E0240 PCR overnight on a gradient 51-56
 * PCR cleaned F2620
 * Set up overnight digests:
 * F2620 (15ul) in buffer 4 cutting Mfe/Xba
 * 3K3 (15ul) in EcoR1 buffer cutting E/X
 * 1AK3 (4ul) in EcoR1 buffer cutting E/X

Sequencing

 * Got back all of the sequencing done july 11, 12
 * However, we realized that we haven't been making glycerols of the things we've sent for sequencing
 * Can't trust the repeating sequences because they may not have been done from the same colony and may not be able to use that colony in the future
 * Made overnights of every construct that looks to be the right size
 * They should all be constructed, scarred, and in the right plasmid
 * E0240-1AK3 from 7/8/07 colonies #1 and #2
 * E0240-3K3 from 7/13/07 colony #9
 * I2047-3K3 from 7/11 #2 and #4
 * I2055-3K3 from 7/13 #3
 * I2055-1AK3 from 7/13 #4 and #16

I2055

 * Ligated I2055 (that contains pcr'ed in promoter and was digested m/n) and 3K3
 * Transformed 3ul of ligation and plated

Primers

 * Ordered new I2055_BB_F and I2056_BB_F primers
 * Spacing was off between the anticipated RBS site and the start of GFP
 * I2055-F 8mer Xba1 ATGCGTAAAGGAGAAGAACTTTTC 52.4
 * CTTAGTAG TCTAGA TGCGTAAAGGAGAAGAACTTTTC


 * I2055-R 8mer xba1 NOT1 ecor1 GTGCTCAGTATCTCTATCACTGATAGG 52.0
 * TCAGCGAT CTCTAGAAGCGGCCGCGAATTC GTGCTCAGTATCTCTATCACTGATAGG


 * I2056-F 8mer Xba1 ATGGCTTCCTCCGAAGACG 54.0
 * CTTAGTAG TCTAGA TGGCTTCCTCCGAAGACG


 * I2056-R same as I2055-R