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

Notebook > AND Gate 1 > Core > Molecular cloning: lacP+SupD/GFP, Sal+SupD/GFP

Molecular cloning: lacP+SupD/GFP, Sal+SupD/GFP
Parts: lacP (R0010)+SupD-term (K228001+B0015)=lacP-SupD-term (K228822) lacP (R0010)+GFP (E0840)=lacP-GFP (K228821) Sal+SupD-term (K228001+B0015)=Sal-SupD-term Sal+GFP (E0840)=Sal-GFP

Resource:  lacP: from parts, myself, SupD-term: from Lin Min, has digested by EcoR1 and Xba1, GFP: from Lin Min, has digested by EcoR1 and Xba1; Sal: from a plasmid from Lin Min;

2009.8.17
Plasmid mini prep: lacP

Double digest:  LacP: 37 ℃ 4 hour

PCR: (sal)

Gel electrophoresis: Products of double digest of L and PCR Marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb Loading buffer and DNA dye: 6× Voltage and time: 60V 5min; 120V 15min Lane 1&2: Sal, PCR product, Lane 3: Marker, Lane 4: lacP, digest product

Sal should be about 1.4kb, and the result is correct! After digested, lacP should have a 200bp one, but I can not see it. So I decided to repeat to amplify the Ecoli and mini prep.

DNA Gel purification: Sal

Double digest:  Sal: 37 ℃ overnight

2009.8.18
PCR product purification: Sal

DNA ligation:  16℃ 4 hour Insert: Sal; Vector: GFP and SupD

Transformation:  Products of ligation (Sal-SupD, Sal-GFP), competent cells 50uL each, Smear to LB plate with Amp

Plasmid mini prep: (again) lacP

Double digest: (again) LacP: 37 ℃ overnight

2009.8.19
Gel electrophoresis: Products of double digest of lacP, Marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb Loading buffer and DNA dye: 6× Voltage and time: 60V 5min; 120V 15min Lane1: lacP: insert 200bp; Lane2: Marker We can find that the smallest but bright one may be RNA, and a little larger and dim one is the insert.

DNA Gel purification:  Insert of lacP

DNA ligation:  16℃ 4 hour Insert: lacP; Vector: GFP and SupD

Transformation: Products of ligation (lacP-SupD, lacP-GFP), competent cells 50uL each, Smear to LB plate with Amp

PCR: (colony PCR to check the Sal-GFP and Sal-SupD)

Double digest: (prepare lacP as a vector) LacP: 37 ℃ 4 hour

Gel electrophoresis: Products of double digest of lacP, and PCR Sal Marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb Loading buffer and DNA dye: 6× Voltage and time: 60V 5min; 120V 15min Lane1~6: Sal-GFP1~6, should be 2.4kb, all are wrong; Lane7~11: Sal-SupD1~5, should be 1.7kb, all are correct; Lane12: negative control; Lane13: Marker; Lane14,15: lacP vector, should be 2.3kb, but there are some pollution plasmids?

2009.8.20
Plasmid mini prep: Sal-SupD

DNA Gel purification:  lacP vector;

The plates of lacP-SupD and lacP-GFP are very good, and the colonies on the lacP-GFP are green. So I do not need to confirm it. PCR: (colony PCR to check the lacP-SupD)

Gel electrophoresis: Products of PCR lacP-SupD Marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb Loading buffer and DNA dye: 6× Voltage and time: 60V 5min; 120V 15min Lane1~4, 6~9: lacP-SupD 1~7; Lane5: Marker; Lane10: negative control; The correct one should about 600bp, and we can find that 1, 2, 3 and 5 are correct.

Result
I successfully constructed the clone: lacP-SupD-term (K228822), lacP-GFP (K2288221) and Sal-SupD-term (which turn out to be wrong after a few days, for more detail, refer to Haoqian Zhang’s notes). I failed to construct Sal-GFP.

Work transfer
Sal transfers to Haoqian Zhang to do, and lacP vector transfers to ShenShan.