Team:PKU Beijing/Notebook/AND Gate 1/Input/LacI TetR 3

From 2009.igem.org

(Difference between revisions)
Lug7 (Talk | contribs)
(New page: {{PKU_Beijing/Header}} {{PKU_Beijing/Sidebar_Notebook}} {{PKU_Beijing/Header2}} ==='''Molecular cloning: Pcat-2M-lacI/tetR-term+lacP/tetP'''=== Parts: K228815/16+R0010/R0040=K228817/18 '...)
Newer edit →

Revision as of 16:40, 1 October 2009

 

Molecular cloning: Pcat-2M-lacI/tetR-term+lacP/tetP

Parts: K228815/16+R0010/R0040=K228817/18

Resource: Pcat-2m-lacI/tetR-term (K228815/16): myself, plasmid, rename as L, T. lacP: part R0010, from He Siheng, already digested; tetP: part R0040, myself, already digested (July 20th)

2009.7.30

Double digest: L, T: Spe1 1uL, Pst1 1uL, plasmid 10uL, Buffer 2uL, water 6uL 37 ℃ 4 hour

Gel electrophoresis: Products of double digest of L, T marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb loading buffer and DNA dye: 6× voltage and time: 60V 5min; 120V 30min lane1: digested product of T; lane2: digested product of L; lane3: marker; PKU 20090730 Shuke Wu 1.JPG The insert of T is about 900bp. The insert of L is about 1.4kb.

DNA Gel purification: Inserts of L, T.

DNA ligation:

System10uL
Insert6uL
vector2uL
water3uL
buffer1uL
T4 DNA ligase1uL

16℃ overnight. Insert: T *2; Vertor: tetP (has already digested by EcoR1 & Xba1)

2009.7.31

Transformation: Products of ligation (T+tetP *2), competent cells 50uL each, Smear to LB plate with Amp

DNA ligation:

System10uL
Insert6uL
vector2uL
water3uL
buffer1uL
T4 DNA ligase1uL

16℃ overnight. Insert: L *2; Vertor: lacP (has already digested by EcoR1 & Xba1, by He Siheng)

Transformation: Products of ligation (L+lacP *2), competent cells 50uL each, Smear to LB plate with Amp

2009.8.1

Every plate is very well: more than 100 clones

PCR: (colony PCR, T-tetP)

System10 uL
Master mix5ul
primer (standard primer)0.5uL each
water4uL template
10 colonies of T-tetP

Gel electrophoresis: (help by Lin Min) Refer to Lin Min’s notes, All of 10 colonies are wrong!!! Repeat!!!

Double digest: (again, tetP) tetP:

EcoR11uL
Xba11uL
plasmid4uL
Buffer2uL
water12uL

37 ℃ 4 hour

Transfer colonies: (L-lacP) Transfer 6 colonies (L-lacP) into 5ml LB, and amplify the Ecoli.

Plasmid mini prep: (L-lacP) 6 colonies of L-lacP

Double digest: (to check the correct L-lacP) 6 L-lacP:

EcoR11uL
Pst11uL
plasmid4uL
Buffer2uL
water12uL

37 ℃ overnight

2009.8.2

Gel electrophoresis: (check the correct L-lacP) Products of double digest Marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb loading buffer and DNA dye: 6× Voltage and time: 60V 5min; 120V 15min PKU 20090802 Shuke Wu 1.JPG I forgot to add the Marker, but from the result we can easily find that all these 6 colonies are wrong!!!

PCR: (colony PCR, L-lacP)

System10 uL
Master mix5ul
primer (standard primer)0.5uL each
water4uL template
24 colonies of L-lacP

Gel electrophoresis: (check the correct L-lacP) 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 PKU 20090802 Shuke Wu 2.JPG From the lane 5 to the last one are 24 results of L-lacP PCR. Since there is not any DNA larger than 1kb, all of these 24 colonies are wrong!!! All these DNA are about 200bp. So they are the result of self link of lacP!!! There must be something wrong with the digested lacP!!!

Double digest: (again, lacP) lacP:

EcoR11uL
Xba11uL
plasmid4uL
Buffer2uL
water12uL

37 ℃ overnight!

DNA ligation (again T+tetP):

System10uL
Insert6uL
vector2uL
water3uL
buffer1uL
T4 DNA ligase1uL

16℃ 4 hours Insert: T *2; Vertor: tetP (digested on Aug.1st)

Transformation: (again T+tetP) Products of ligation (T+tetP *2), competent cells 50uL each, Smear to LB plate with Amp

2009.8.3

PCR product purification: lacP (digested yesterday)

DNA ligation (again L+lacP):

System10uL
Insert6uL
vector2uL
water3uL
buffer1uL
T4 DNA ligase1uL

16℃ 4 hours Insert: L *2; Vertor: lacP (digested on Aug.2nd )

PCR: (colony PCR, T-tetP)

System10 uL
Master mix5ul
primer (standard primer)0.5uL each
water4uL template
10 colonies of T-tetP

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 PKU 20090803 Shuke Wu 1.JPG Since there is not any DNA larger than 1kb, all of these 10 colonies are wrong Again!!! Bad Luck!!!!!

Transformation: (again L+lacP) Products of ligation (L+ lacP *2), competent cells 50uL each, Smear to LB plate with Amp

Double digest: (the 3rd time! tetP and T) tetP:

EcoR11uL
Xba11uL
plasmid4uL
Buffer2uL
water12uL

T:

pe11uL
Pst11uL
plasmid10uL
Buffer2uL
water6uL

37 ℃ overnight!

2009.8.4

PCR product purification: tetP (digested yesterday)

Gel electrophoresis: Products of double digest of L, T marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb loading buffer and DNA dye: 6× voltage and time: 60V 5min; 120V 30min lane1: digested product of T; lane2: marker PKU 20090804 Shuke Wu 1.JPG The insert should be 900 bp, and it is correct!

DNA Gel purification: Insert of T.

DNA ligation (the 3rd time T+tetP):

System10uL
Insert6uL
vector2uL
water3uL
buffer1uL
T4 DNA ligase1uL

16℃ 4 hours Insert: T *2 (new); Vertor: tetP (new);

Transformation: (the 3rd time T+tetP) Products of ligation (T+tetP *2), competent cells 50uL each, Smear to LB plate with Amp

PCR: (the 2nd time colony PCR, L-lacP)

System10 uL
Master mix5ul
primer (standard primer)0.5uL each
water4uL template
12 colonies of L-lacP

Gel electrophoresis: (check the correct L-lacP) 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 PKU 20090804 Shuke Wu 2.JPG The insert of correct L-lacP is about 1.4kb!!! 9 of 12 colonies are correct!!!!!

2009.8.5

PCR: (the 3rd time colony PCR, T-tetP)

System10 uL
Master mix5ul
primer (standard primer)0.5uL each
water4uL template
12 colonies of T-tetP

Gel electrophoresis: (check the correct T-tetP) Products of PCR Marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb loading buffer and DNA dye: 6× Voltage and time: 60V 5min; 120V 60min Lane 1~12: T-tetP 1~12 Lane 13: Marker The insert is about 1kb, and 9 of these 12 colonies are CORRECT!!!! PKU 20090805 Shuke Wu 1.JPG

Result

At last, I successfully constructed: Pcat-2M-lacI/tetR-term+lacP/tetP, and they are the parts K228817/18.

Experience

The vector is very important in this cloning. We should digest completely all the vectors, in order to prevent the self-linkage of the vectors. My experience is that if you want to digest 4ul plasmid as vector, you had batter digest it overnight. If you want to quick such as in two hours, reduce the amount of plasmid.





^Top