Team:Calgary/27 July 2009
From 2009.igem.org
University of Calgary
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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.
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CHINMOYEE
Descriptive Title of What You're Doing
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EMILY
Descriptive Title of What You're Doing
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FAHD
Descriptive Title of What You're Doing
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IMAN
Descriptive Title of What You're Doing
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JAMIE
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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
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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 |
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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 |
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4 |
hold |
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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
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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 |
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Positive control = ∆LuxPQ in psB1AK3
PCR conditions
| # of cycles |
Temp (ºC) |
Time |
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| 1 |
94 |
6 min |
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| 36 |
94 |
30sec |
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| 55 |
45sec |
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| 72 |
6min 20sec |
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| 1 |
72 |
10min |
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4 |
hold |
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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 |
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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 |
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| 2μL
REact 4 |
2μL REact 4 |
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| 0.75
μL SpeI |
0.75 μL SpeI |
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| 0.75
μL PstI |
0.75 μL PstI |
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| 2
μL of ∆LuxPQ in AK3 C2 [112.6ng/μL] |
2 μL of ∆LuxPQ in AK3
C5 [115.3ng/μL] |
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| 14.5
μL ddH2O |
14.5 μL ddH2O |
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Insert
| 2μL
REact 2 |
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| 0.75
μL XbaI |
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| 0.75
μL PstI |
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| 2
μL of LuxPQ-B0015-R0040-LuxOU-B0015 in AK3 C1 [156.3ng/μL] |
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| 12.5
μL ddH2O |
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Put in the incubator at 37ºC overnight.
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KATIE
Descriptive Title of What You're Doing
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KEVIN
Descriptive of What You're Doing
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MANDY
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PATRICK
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PRIMA
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STEFAN
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VICKI
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