Jessica Karen Wong/Notebook/2007-7-16

T9002 Colony PCR Gel

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

PCR of 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