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Team:Calgary/27 July 2009
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| - | + | Colony PCR of LuxPQ-B0015-R0040-LuxOU-B0015 construct in psB1AC3 using BBK CP F/R and LuxPQ F/LuxOU R primers | |
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<div class="desc"> | <div class="desc"> | ||
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| - | + | Purpose: To verify the presence of LuxPQ-B0015-R0040-LuxOU-B0015 construct in psB1AC3. | |
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| + | 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 | ||
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| + | PCR conditions | ||
| + | # of cycles Temp (ºC) Time | ||
| + | 1 94 6 min | ||
| + | 36 94 30sec | ||
| + | 55 45sec | ||
| + | 72 6min 20sec | ||
| + | 1 72 10min | ||
| + | 4 hold | ||
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| + | Result: the only bands that appeared on the gel were those of the ladders. Therefore, start new construction. | ||
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| + | 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 | ||
| + | |||
| + | |||
| + | Result: Of the 6 colonies, 5 had bands of the correct size (~3.9kb). | ||
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| + | Isolating plasmid from LuxPQ-B0015-R0040-LuxOU-B0015 (AC) and LuxPQ (in AC) | ||
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| + | Purpose: isolate and measure concentrations of pure plasmids. | ||
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| + | 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 1.80 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 1.70 2.27 16.3 | ||
| + | PQ-B-R-OU-B in AC3 C4 1.71 1.79 17.0 | ||
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| + | Construction of LuxPQ-B0015-R0040-LuxOU-B0015 by inserting B0015-R0040-LuxOU-B0015 (AK) into LuxPQ (in AC) | ||
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| + | 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 | ||
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| + | Put in the incubator at 37ºC overnight. | ||
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Revision as of 22:18, 28 July 2009
UNIVERSITY OF CALGARY
