Week3 6/27/10-7/3/10

From 2010.igem.org

(Difference between revisions)
(New page: ===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 we...)
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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.
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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----
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===7/1/10===
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First weekly meeting with Professor Jewett, Leonard, and Mordacq.
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 +
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.
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 +
Kit to Stock:
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*'''1-12O''' (e0840: rbs30-gfp-2xterm)
 +
*'''1-12M''' (e0240: rbs32-gfp-2xterm)
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*'''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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 +
----
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===7/3/10===
 +
Miniprep -> ~50ng/μl
 +
 +
Called Avi about kit to stock protocol - "18hr way too long"
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 +
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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Revision as of 17:53, 16 October 2010

Contents

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).


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).


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.


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).


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).


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).