Team:Calgary/27 July 2009

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University of Calgary

UNIVERSITY OF CALGARY



THIS MONTH

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NOTEBOOK PAGE INDEX
Carol
Chinmoyee
Emily
Fahd
Iman
Jamie
Jeremy
Katie
Kevin
Mandy
Patrick
Prima
Stefan
Vicki



CALENDAR

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JULY 27, 2009


CAROL

Finishing Modeling Paper

  • Finished writing modeling paper that summarizes what we will be doing once the circuit is completed. The experiments should not take long to accomplished and these results can be tabulated in Matlab quickly.
  • Plated the week-long ligation of the promoter library in both XL Gold Ultra competent cells and Top 10 cells, but there is no growth.
  • A reason that we thought of that might have caused minimal colonies is the phosphatase step. The 32 primers that we have synthesized does not have 5' phosphates. If we phosphatase the vector, that might result in no ligation.
  • Re-started restriction enzyme digestion on vector pCS26 with XhoI and BamHI at 37oC for 4 hours. Then I ligated the promoter with the vector with Quick Ligase for 5 minutes. Then I transformed the plasmids into both XL Gold Ultracompetent cells and Top 10 cells. Incubate at 37oC for 16 hours.


CHINMOYEE

Descriptive Title of What You're Doing

WIKI CODING HERE


EMILY

Construction of J13002-LuxOD47E-B0015

  • Did a construction digest of the J13002-LuxOD47E construct and B0015 terminator with XbaI/ PstI/ SpeI enzymes.
  • Transformation into TOP10 cells and plated on AC plates.
  • Hopefully things will go smoothly from here and my circuit will be fully-contructed by Thursday!

Ethics/ Outreach

  • Today I talked with Fahd, Stefan and Mandy about our Ethics paper as well as our conference. Mandy has a location in mind and she want Fahd and I to log into Second Life and explore the ethics area a bit. Mandy had a chance to look at our paper first thing this morning and she made some good suggestions for areas to add on to the introduction, bringing in some of the stuff that we learned about in the webinar. I spent a large part of today re-writing that part and I will have a copy of the rough draft of our paper by tonight!



FAHD

Descriptive Title of What You're Doing

WIKI CODING HERE


IMAN

Descriptive Title of What You're Doing

WIKI CODING HERE


JAMIE

Descriptive Title of What You're Doing

WIKI CODING HERE


JEREMY

Colony PCR of LuxPQ-B0015-R0040-LuxOU-B0015 construct in psB1AC3 using BBK CP F/R and LuxPQ F/LuxOU R primers

Colony PCR of LuxPQ-B0015-R0040-LuxOU-B0015 construct in psB1AC3 using BBK CP F/R and LuxPQ F/LuxOU R primers
Purpose: To verify the presence of LuxPQ-B0015-R0040-LuxOU-B0015 construct in psB1AC3.

Protocol
  MM1 (6x) (μL) MM2 (6x) (μL)
10X PCR buffer minus MgCl2 30 30
10mM dNTPs 6 6
50mM MgCl2 9 9
F primer 6 (BBK CP F) 6 (LuxPQ F)
R primer 6 (BBK CP R) 6 (LuxOU R)
ddH2O 241.8 241.8
pTaq 1.2 1.2


Positive control = LuxPQ-B0015-R0040-LuxOU-B0015 in AK3

PCR conditions

# of cycles Temp (ºC) Time
1 94 6 min
36 94 30sec
55 45sec
72 6min 20sec
1 72 10min
  4 hold

Result: the only bands that appeared on the gel were those of the ladders. Therefore, start new construction.




Colony PCR of ∆LuxPQ in psB1AC3 using BBK CP F/R and LuxPQ F/R primers
Purpose: To verify the presence of ∆LuxPQ in psB1AC3 (to verify a successful plasmid switch)
Protocol

  MM1 (8x) (μL) MM2 (6x) (μL)
10X PCR buffer minus MgCl2 40 40
10mM dNTPs 8 8
50mM MgCl2 12 12
F primer 8 (LuxPQ F) 8 (BBK CP F)
R primer 8 (LuxPQ R) 8 (BBK CP R)
ddH2O 322.4 322.4
pTaq 1.6 1.6

Positive control = ∆LuxPQ in psB1AK3

PCR conditions
# of cycles Temp (ºC) Time
1 94 6 min
36 94 30sec
55 45sec
72 6min 20sec
1 72 10min
  4 hold


Calgary PQplasmid switch AC.png


Isolating plasmid from LuxPQ-B0015-R0040-LuxOU-B0015 (AC) and LuxPQ (in AC)
Purpose: isolate and measure concentrations of pure plasmids.

DNA 260/280 260/230 Conc. [ng/μL]
∆LuxPQ in AC3 C1 1.83 2.23 146.8
∆LuxPQ in AC3 C2 1.81 2.19 112.6
∆LuxPQ in AC3 C3 1.8 2.27 145.8
∆LuxPQ in AC3 C4 1.78 2.09 96.6
∆LuxPQ in AC3 C5 1.81 2.25 115.3
∆LuxPQ in AC3 C6 1.79 2.17 44.8
PQ-B-R-OU-B in AC3 C1 1.76 1.86 29.4
PQ-B-R-OU-B in AC3 C2 1.59 1.44 20.1
PQ-B-R-OU-B in AC3 C3 1.7 2.27 16.3
PQ-B-R-OU-B in AC3 C4 1.71 1.79 17




Construction of LuxPQ-B0015-R0040-LuxOU-B0015 by inserting B0015-R0040-LuxOU-B0015 (AK) into LuxPQ (in AC)

Purpose: To construct LuxPQ-B0015-R0040-LuxOU-B0015 by inserting B0015-R0040-LuxOU-B0015 (AK) into LuxPQ (in AC)
Protocol
Recipient

Recipient 1 Recipient 2
2μL REact 4 2μL REact 4
0.75 μL SpeI 0.75 μL SpeI
0.75 μL PstI 0.75 μL PstI
2 μL of ∆LuxPQ in AK3 C2 [112.6ng/μL] 2 μL of ∆LuxPQ in AK3 C5 [115.3ng/μL]
14.5 μL ddH2O 14.5 μL ddH2O


Insert
2μL REact 2
0.75 μL XbaI
0.75 μL PstI
2 μL of LuxPQ-B0015-R0040-LuxOU-B0015 in AK3 C1 [156.3ng/μL]
12.5 μL ddH2O


Put in the incubator at 37ºC overnight.



KATIE

Descriptive Title of What You're Doing

WIKI CODING HERE


KEVIN

Descriptive of What You're Doing

WIKI CODING HERE


MANDY

Descriptive Title of What You're Doing

WIKI CODING HERE


PATRICK

Descriptive Title of What You're Doing

WIKI CODING HERE


PRIMA

Descriptive Title of What You're Doing

WIKI CODING HERE


STEFAN

Descriptive Title of What You're Doing

WIKI CODING HERE


VICKI

Descriptive Title of What You're Doing

WIKI CODING HERE

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