Team:PKU Beijing/Notebook/AND Gate 1/Core/Shuke Wu 3

Molecular cloning: AND gate LSAT*9

My LAST CLONING this summer!!

Parts:
lacP-SupD (K228822)+araC-T7ptag=lacP-SupD-AraO-T7ptag, LSAT, (K228823~31)

Resource:
lacP-SupD: plasmid, from myself; renamed as LS;
araC-T7ptag: insert (digested by Xba1 and Pst1), from GuoSheng Zhang.

2009.8.23

Plasmid mini prep:
LG*3, LS*3;

Double digest:
LG1, 2 and 3:

LS1, 2 and 3:

37 ℃ 4 hour

Gel electrophoresis:
Products of double digest of LG and LS,
marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb
loading buffer and DNA dye: 6×
voltage and time: 60V 5min; 120V 15min

lane1~3: LG1~3, should has insert 1.1k and vector 3.4k, all are correct!!
Lane5~7: LS1~3, should be 4.1k, all are correct.
Lane4: marker;

DNA Gel purification:
LS1, 2 and 3;

DNA ligation:

16℃ 4 hour
Insert: araC-T7ptag*9;
Vertor: LS1;

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

2009.8.24

Every plate is not very good: less than ten clones each plate; and LSAT-2G plate has no clone!!

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 30min

lane1~4: LSAT-2M (2M means the RBS);
lane5: 5J;
lane6, 7: Marker
lane8: 5N;
lane9, 10: 1H;
lane11, 12: 2k;
lane13: 11N;
lane14~17: 2I;
lane18+under row lane1, 2, 5: 1J;
Under row lane3, 4: Marker
Under row lane6: negative control;

If the colony is correct, its insert should be 4.6kb. So we can find only 2I and 1J are correct! REPEAT!!
Because the insert is large, sometimes can not be amplified by taq enzyme, I decided to mini prep some of them.
Transfer the colonies: 2M(2), 5J, 5N, 1H, 2K, 2I(1, 3), 1J(3, 4) into 5 mL LB each.

Double digest (2nd time):
LS1:

37 ℃ overnight

2009.8.25

Plasmid mini prep:
2M(2), 5J, 5N, 1H, 2K, 2I(1, 3), 1J(3, 4);

Digest:
Single digest: 2M(2), 5J, 5N, 1H, 2K, 2I(1, 3), 1J(3, 4):

Double digest: 2M(2); 2I(1, 3); 1J (3, 4):

37 ℃ 4 hour

Gel electrophoresis:
Products of digest
Marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb
loading buffer and DNA dye: 6×
Voltage and time: 60V 5min; 120V 30min
Lane1: lacP-SupD digested by Spe1 and Pst1
Lane2~5: Single digest:, 5J, 5N, 1H, 2K,
Lane6, 12: Marker;
Lane7~11: Double digest: 2M(2); 2I(1, 3); 1J (3, 4);
Lane13~17: single digest: 2M(2); 2I(1, 3); 1J (3, 4);

Correct colony should have 3.4k vector and 4.6k insert, and the whole plasmid is 8k. And from the gel: we found that only 2I(1, 3), 1J (3, 4) are correct, which consistent with the result of PCR.

DNA Gel purification: (2nd time)
lacP-supD (LS1),

DNA ligation: (2nd time)

16℃ 4 hour
Insert: araC-T7ptag*7 (without 2I and 1J);
Vertor: LS1;

Transformation: (2nd time) (Helped by Haoqian Zhang)
Products of ligation, competent cells 50uL each,
Smear to LB plate with Kan.

2009.8.26

Every plate is not very good, but better than the first time: more than ten clones each plate.

PCR: (colony PCR) (2nd time)

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 30min

Lane1~3: LSAT-11N
Lane4~6: 5N
Lane7~9: 1H
Lane10~12: 5J
Lane13: Marker
Lane14~16: 2K
Lane17~19: 2G
Lane20~24: 2M
Lane25: PCR negative control
We can find that every clone has some correct one.

Transfer the colonies:11N-2, 5N-1, 1H-2, 5J-2, 2K-3, 2G-3, 2M-3,4,5 into 5 mL LB each.

2009.8.27

Plasmid mini prep: (2nd time)
11N-2, 5N-1, 1H-2, 5J-2, 2K-3, 2G-3, 2M-3,4,5

Digest: (2nd time)
Single digest: 11N-2, 5N-1, 1H-2, 5J-2, 2K-3, 2G-3, 2M-3,4,5:

Double digest: 211N-2, 5N-1, 1H-2, 5J-2, 2K-3, 2G-3, 2M-3,4,5:

37 ℃ 4 hour

Gel electrophoresis:
Products of digest
Marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb
loading buffer and DNA dye: 6×
Voltage and time: 60V 5min; 120V 30min
Lane 1~9: Single digest: 11N-2, 5N-1, 1H-2, 5J-2, 2K-3, 2G-3, 2M-3,4,5
Lane 10: Marker
Lane 11~18: Double digest: 211N-2, 5N-1, 1H-2, 5J-2, 2K-3, 2G-3, 2M-3,4
Correct colony should have 3.4k vector and 4.6k insert, and the whole plasmid is 8k. And from the gel: we found that all of they are CORRECT!!!

Result

I have already successfully constructed all the night AND gates: LSAT lacP-SupD-AraC-T7ptag, and they are parts K228823~31.
Now I finished all the cloning!!!
Appreciate every member of our iGEM group!!!
Go on to test my AND gates

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