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 precipiation (RESULTS)
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