Team:Duke/Notebook/August

The two types of cells to make competent were DH5-aZ1 cells containing the reporter gene and cells with the reporter and GFP1 site. Charlie started cultures on Saturday evening from a plate into LB + antibiotics. The antibiotics used were ampicillin in the reporter-only cells and ampicillin plus chloramphenicol in the reporter/GFP1 cells. On Sunday morning, he put them in the cold room and then took the OD that evening by adding 100 uL of culture to 900 uL broth plus antibiotics:

• R: 0.276
• R+G1: 0.268

He then started four cultures by adding 120 mL LB/appropriate antibiotic(s) to the following and growing them at 24 C:

• 70 uL R
• 0.7 mL R (denser culture)
• 70 uL R+G1
• 0.7 mL R+G1 (denser culture)

Today, at 10:25 AM, OD was measured again, but undiluted.

• dense R: 0.023
• R: -0.001
• dense R+G1: 0.0037
• R+G1: -0.000

Measurements were repeated at 12:34 PM, but only on the denser cultures.

• dense R: 0.038
• dense R+G1: 0.061

The temperature was increased to improve doubling time.

Redo minipreps from yesterday - culture tubes grew overnight from salvaged cells

• pSB1C3-dCas9-tracrRNA - 171.3 ng/ul
• pSB3C5-R0040-a-tracr 1 - 257.5 ng/ul
• pSB3C5-R0040-a-tracr 2 - 202.7 ng/ul
• pSB3C5-R0040-a-tracr 3 - 200.5 ng/ul
• pSB3C5-R0040-a-tracr 4 - 198.3 ng/ul

Analytical digest of Tube 1 (pSB1C3-dCas9-tracrRNA) using XhoI and BamHI

• 2 hour digest at 37 degrees C
• 3X Master Mix:
• 3 ul Cutsmart
• 22.5 ul water
• 0.75 ul XhoI
• 0.75 ul BamHI
• 9 ul Master Mix added to 1 ul of pdCas9 in one tube and 1 ul of pSB1C3-dCas9-tracrRNA in another tube
• Gel results were consistent with an unsuccessful ligation. 3 bands in the experimental column (4kb, 1.8 kb, and 0.9 kb) were expected. The results were more consistent with pSB1C3 and no insert. The band size in the pdCas9 control lane also looked wrong because it was too big. The pdCas9 tube may have been faulty or the BamHI might have been faulty.