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From 2010.igem.org

__NOTPC__

Contents 1 6/28/10 2 6/29/10 3 6/30/10 4 7/1/10 5 7/2/10 6 7/3/10

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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