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From 2010.igem.org

Week3 Highlight

We re-extracted 12O and 6G and extracted 3-20M. We also met with Dr. Russin about the microscopy aspect of our project. We plan on focusing on confocal microscopy to view the cross section of our biofilm. We established weekly meetings with professor Jewett, Leonard, and Mordacq. We began PCRing CHS3 using yeast genome. We also spoke to Avi about our miniprep problems and he advised us that 18 hours of incubation was "way too long".

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6/28/10

Kit to Stock

• 1-12O (e0840: rbs30-gfp-2xterm)

• 1-6G (r0011: inducible promoter)

• 3-20M (K124017: Bacteriophage Lysis Cassette S105, R, and Rz)

Plates were incubated at 37°C overnight (starting at 2:30 pm).

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6/29/10

Selected single 1-12O, 1-6G, 3-20M colonies and grew them in Amp-LB broth for 18 hours (starting at 4:15 pm).

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6/30/10

Poured 2 Sleeves of Kan and Amp plates respectively

Met with Dr. Russin (BIF)

Sent emails regarding biofilm and biofilm imaging: Aaron Packman, Wei Zhang, Kimberly Grey

Most likely going to use water immersion Leica (Confocal Microscope) + low MP agar (enrobing technique)

Red colonies reappeared on 1-12O plates; Fluorescent Microscopy revealed negligible activity; still unsure why colonies are red.

Miniprepped 1-12O, 3-20M, 1-6G: nanodrop -> mostly below 20ng, 1 above 100ng; stored in -20°C

Re-miniprepped after growing in liquid LB for 3 hours; nanodrop -> around 50ng/μL, 2 around 100ng/μL; stored in -20°C

Transferred overnight cultures into new 5ml LB broths. Incubated overnight in an angled shaker (starting from 4:20pm)

Plated 10μl, 25μl, 100μl, and 200μl (added LB up to 200μL) of the KR 1-12O overnight culture. Intend to measure the thickness of E.Coli lawn.

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7/1/10

First weekly meeting with Professor Jewett, Leonard, and Mordacq.

Removed overnight cultures from incubator at 10:00am. Miniprep (let the elution buffer sit for 10 minutes with the DNA before centrifuging) --> 1 around 30ng/μL, mostly around 75ng/μL.

Started PCR of the chitin synthase gene using yeast genome.

Kit to Stock:

• 1-12O (e0840: rbs30-gfp-2xterm)
• 1-12M (e0240: rbs32-gfp-2xterm)
• 1-6G (r0011: inducible promoter)

Plates were incubated at 37°C overnight (starting at 5pm).

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7/2/10

Removed overnight plates from 37°C at 10:00am and moved them to the 4°C cold room.

Gel extraction on Chitin Synthase PCR product.

Colonies were selected from the NB-12M, NB-6G, NB-12O, T10-12M, T10-6G, and T10-12O plates and transferred to LB broth and incubated for 18 hours at 37°C (starting at 4:00pm).

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7/3/10

Miniprep -> ~50ng/μl

Called Avi about kit to stock protocol - "18hr way too long"

2 Sets of colonies were selected from the NB-12M, NB-6G, NB-12O, T10-12M, T10-6G, and T10-12O plates and transferred to LB broth and incubated for 13 and 15 hours at 37°C (starting at 9:15pm).

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