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
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
Positive control = ∆LuxPQ in psB1AK3
PCR conditions
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.
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
Insert
Put in the incubator at 37ºC overnight.