Notebook > Assembly > Assembling : bistable-> pSB1A2 & pSB6A1
Assembling : bistable-> pSB1A2 & pSB6A1
Resource
Bistable, pSB1A2 and pSB6A1 form Min Lin.
2009.10.1
Plasmid mini prep
Bistable, pSB1A2, pSB6A1;
Double digest
Bistable, pSB1A2, pSB6A1: Pst1 1uL, Xba1 1uL, plasmid 10uL, Buffer 2uL, water 6uL
37 ℃ 4 hour
Gel electrophoresis
Products of double digest.
Separate the inserts and vectors.
DNA Gel purification
Insert of bistable;
Vectors of pSB1A2 and pSB6A1;
DNA ligation
System 10uL: Insert 6uL, vector 2uL, buffer 1uL, T4 DNA ligase 1uL
16℃ 2 hour
Transformation
Products of ligation (bistable-pSB1A2, bistable-pSB6A1), competent cells 50uL each,
Smear to LB plate with Amp
2009.10.2
Result
There are more than 100 colonies on each plate. I picked 6 of them to liquid LB to cultivate overnight to confirm the clonings.
2009.10.3
Plasmid mini prep:
Bistable on pSB1A2 (6 different clonings);
Bistable on pSB6A1 (6 different clonings);
Double digest: to check the results
Bistable on pSB1A2 & pSB6A1: Pst1 1uL, Xba1 1uL, plasmid 4uL, Buffer 2uL, water 12uL
37 ℃ 2 hour
Gel electrophoresis: to confirm
Products of double digest.
Separate the inserts and vectors.
Function
Most of colonies on the cloning plates became green on Oct. 3rd, which means the insert is Bistable, because the Bistable is on the CI434 side is green. This phenomenon proved that the clonings: transfer Bistable to pSB1A2 and pSB6A1 are successful.
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