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
Result: Of the 6 colonies, 5 had bands of the correct size (~3.9kb).
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 1.80 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
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.
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