Team:Northwestern/08 10

Notebook

Wednesday, August 10th

Results:

Cam Plate Test

All concentrations of Cam showed colonies. The 12.5 ug/mL plates showed more colonies, which is to be expected because it was the lowest concentration of antibiotic used. We used the high copy number cells from streak of Patrick’s cells on 08.09.16.

Figure 1: Cam 12.5 ug/mL plate 1

Figure 2: Cam 12.5 ug/mL plate 2

Figure 3: Cam 25 ug/mL plate 1

Figure 4: Cam 25 ug/mL plate 2

Figure 5: Cam 20 ug/mL plate 1

Figure 6: Cam 20 ug/mL plate 2

Figure 7: Cam 30 ug/mL plate 1

Figure 8: Cam 30 ug/mL plate 2

Cas9 Sequencing

Looks like “old” Cas9 1:3 worked and is assembled. Will now transform miniprepped probably ok DNA and plate on different antibiotic concentration.

Tasks:

Jordan

  • Gave check to ChemE office
  • Transformed assembled Cas9 miniprepped plasmids into cells
    • Transformed 1 uL (130 ng) of Cas9 miniprepped on 8/9 from culture with chloramphenicol, 1 ul (240 ng) of Cas9 miniprepped on 8/7 from no antibiotic culture
    • Controls:
      • positive—J04500 in SB1C3 (RFP), 20 pg in 1 uL
      • negative—1 ul nuclease free water from freezer aliquot
    • Conditions:
      • Competent cells were retrieved from -80 about 15 minutes before use
      • On ice for 30 minutes
      • Transformed for 45 seconds- 42 degrees confirmed on thermometer
      • On ice for 5 minutes
      • 200 uL of SOC added from Patrick’s aliquot- topped flamed before use
      • Incubated 1 hour
      • Plated 250 uL of each

Paul

  • Got sequencing results back
  • Talked with Kelly, Patrick about our cells’ transformation efficiency

Sam

  • Made UNSW meeting doc & worked on content
  • Researched JC8031 to make sure we know why it works
  • Worked with Jordan to get funds settled from Eligo

Shu

  • Dpn1 Digest of linearized mRFP backbone—0.5 µL per 50 µL reaction
  • Ran two gels

Tyler

  • Ran gel of mRFP Linearization before and after miniprep
  • m1 and m2 were prepared from 2 different colonies. The negative control had DNA, and 200 ng of backbone was used in this reaction. This gel was thrown out.
  • This was a test to see whether the miniprep was contaminated. It was not.
  • E-mailed Jarod about plates/tips
  • Ordered another sequencing primer to better sequence our Cas9 part