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From 2010.igem.org

Week4 Highlights

We changed our overnight culture protocol to 13 hours instead of 18 hours, but we still have problems with low DNA yields. We thought maybe our antibiotic levels were to blame. Our first PCR reaction of CHS3 failed. We met with Peggy Saks and learned about the Keio Collection where we can find ChiA and ydgG knockouts. We also purchased our IPTG spray bottles.

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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)

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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)

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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).

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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)

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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

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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.

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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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