Week4 7/4/10-7/10/10

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7/4/10

13hr(2ml) -> miniprep -> ~45ng/μl

13hr(3ml) -> miniprep -> ~55ng/μl

15hr(2ml) -> miniprep -> 40ng/μl

Also, NB consistently gave higher yields than T10 by 5~10ng/μl

Note: 18hr gave higher yields than either 13hr or 15hr

Protocol: 18hr, NB, 5ml incubation (spin all down)

7/5/10

CHS3 Gel Extraction -> 8ng/μl

Restarted CHS3 PCR added 5 cycles.

Kit to Stock:

  • 2-8E (j06702: RFP)
  • 1-18C (j23100: CP1)
  • 1-18K (j23104: CP2)
  • 1-18M (j23105: CP3)
  • 1-2M (b0034: RBS1)
  • 1-2G (b0031: RBS2)
  • 1-2I (b0032: RBS3)
  • 1-23L(b0015: DT1)
  • 1-2O (c0012: LacL)

Reincubated (5μl transfer to liquid culture) 1-12O, 1-12M, 1-6G (GFP1, GFP2, LacP1 respectively)

Notes: Need Cells; Reorganize Lab (assign benches)

7/6/10

Miniprep (16hr/5ml) -> around 50ng/μl

Ran another gel extraction for CHS3

Kit to Stock:

  • 1-1D (r0010: LacP2)
  • 3-20K (K124014: Holin2)

Plated 1-1D, 3-20K and incubated overnight.

Colonies were selected from the NB-8E, NB-18C, NB-18K, NB-18M, NB-2M, NB-2G, T10-2I, T10-23L, T10-6K, and T10-2O plates and transferred to LB broth and incubated for 16 hours at 37°C (starting at 6:00pm).

7/7/10

Miniprep (25ul of Amp/5ml spin down) --> 3 ~100ng/μl and 2 ~180ng/μl

Miniprep (50ul of Amp/1.5ml spin down) --> all ~20ng/μl

Miniprep (50ul of Amp/3.5ml spin down) --> all ~20ng/μl

50μg/ml of Amp = seems to result in more DNA (half the concentration of what we previously used)

Gel extraction --> very bright CHS3 band (10.1ng/μl)

Colonies were selected from the T10-2I, T10-23L, T10-6K, T10-2O, T10-1D, T10-20K plates and transferred to LB broth and incubated for 16 hours at 37°C (starting at 7:30pm)

7/8/10

Ethanol precipitation of 1-6G, 2-8E, 1-12O, and 1-12M

Keio Collection: ChiA knockouts grew, but YdgG knockouts did not (possibly dead due to storage in -20 instead of -80)

Started another PCR reaction of CHS3

Miniprep (5ml spin down/25ul of antibiotic) --> mostly ~50ng/ul

Miniprep (5ml spin down/20ul of antibiotic) --> mostly ~70ng/ul

Diagrammed out our assembly methods for the CHS3 plasmid and the Apoptotic plasmid


7/9/10

Finished the ethanol precipitation

Started assembling the LacP-RFP, LacP-GFP1, and LacP-GFP2 pieces for use in testing the Lac promoter.

7/10/10

IPTG Spray Bottles: 10 Sprays = ~1.25ml --> .125ml/spray 100mM IPTG Stpcl = 1 gram/41.96ml

3 Sprays of 1mM IPTG (started at 5:15pm)

  • 30 Min
  • 1 Hour

3 Sprays of 5mM IPTG (started at 5:15pm)

  • 30 Min
  • 1 Hour
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