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

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.

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