Team:PKU Beijing/Notebook/AND Gate 1/Core/Shuke Wu 2

Notebook > AND Gate 1 > Core > Transfer backbone: lacP+SupD/GFP to low copy pSB4K5

Transfer backbone: lacP+SupD/GFP to low copy pSB4K5
Motivation: In order to make use of lacIQ of the F plasmid of JM109 (more detail refer to my notes 0817), the AND gates should be build on low copy plasmid. And transfer at this time can save a lot of workload.

Resource:  lacP-SupD-term (K228822): plasmid, from myself; renamed as LS; lacP-GFP (K228821): plasmid, from myself; LG; low copy backbone, pSB4K5: vector, has already digested by EcoR1 and Pst1, from ShenShan.

2009.8.21
Plasmid mini prep: LG, LS1, LS2;

Double digest:  LG, LS1, LS2: 37 ℃ 4 hour

Gel electrophoresis: Products of double digest of LG, LS1 and LS2; marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb loading buffer and DNA dye: 6× voltage and time: 60V 5min; 120V 15min lane1: LG; lane3,4: LS1, LS2; lane2: marker; The insert of LG is 1.1k, correct; The insert of LS is 600bp, correct;

DNA Gel purification: Insert of LG, LS1 and LS2.

DNA ligation:  16℃ 4 hour Insert: LG, LS1 and LS2; Vertor: pSB4K5

Transformation:  Products of ligation, competent cells 50uL each, Smear to LB plate with Kan.

2009.8.22
PCR: (colony PCR)

Gel electrophoresis: Products of PCR Marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb loading buffer and DNA dye: 6× Voltage and time: 60V 5min; 120V 15min Lane1~5: LG1~5; Lane6: PCR negative control; Lane7: Marker Lane8~13: LS1~6 All colonies are correct!!!

Result
I successfully transferred lacP-SupD-term (K228822) and lacP-GFP (K228821) to low copy plasmid pSB4K5.