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

From 2009.igem.org

(Difference between revisions)
(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 '...)
 
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{{PKU_Beijing/Sidebar_Notebook}}
{{PKU_Beijing/Sidebar_Notebook}}
{{PKU_Beijing/Header2}}
{{PKU_Beijing/Header2}}
 +
[[Team:PKU_Beijing/Notebook|Notebook]] > [[Team:PKU_Beijing/Notebook/AND_Gate_1|AND Gate 1]] > [[Team:PKU_Beijing/Notebook/AND_Gate_1/Input|Input]] > [[Team:PKU_Beijing/Notebook/AND_Gate_1/Input/LacI_TetR_3|Molecular cloning: Pcat-2M-lacI/tetR-term+lacP/tetP]]
==='''Molecular cloning: Pcat-2M-lacI/tetR-term+lacP/tetP'''===  
==='''Molecular cloning: Pcat-2M-lacI/tetR-term+lacP/tetP'''===  
 +
Parts: K228815/16+R0010/R0040=K228817/18
Parts: K228815/16+R0010/R0040=K228817/18
-
'''Resource:'''  
+
'''Resource:'''<br>
-
Pcat-2m-lacI/tetR-term (K228815/16): myself, plasmid, rename as L, T.  
+
Pcat-2m-lacI/tetR-term (K228815/16): myself, plasmid, rename as L, T. <br>
-
lacP: part R0010, from He Siheng, already digested;   
+
lacP: part R0010, from He Siheng, already digested;  <br>
tetP: part R0040, myself, already digested (July 20th)
tetP: part R0040, myself, already digested (July 20th)
==='''2009.7.30'''===
==='''2009.7.30'''===
   
   
-
'''Double digest:'''
+
'''Double digest:'''<br>
-
L, T: Spe1 1uL, Pst1 1uL, plasmid 10uL, Buffer 2uL, water 6uL
+
L, T:  
 +
{|cellpadding=5
 +
|Spe1||1uL
 +
|-
 +
|Pst1||1uL
 +
|-
 +
|plasmid||10uL
 +
|-
 +
|Buffer||2uL
 +
|-
 +
|water||6uL
 +
|}
37 ℃ 4 hour
37 ℃ 4 hour
-
'''Gel electrophoresis:'''
+
'''Gel electrophoresis:'''<br>
-
Products of double digest of L, T  
+
Products of double digest of L, T <br>
-
marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb
+
marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb<br>
-
loading buffer and DNA dye: 6×
+
loading buffer and DNA dye: 6×<br>
-
voltage and time: 60V 5min; 120V 30min
+
voltage and time: 60V 5min; 120V 30min<br>
-
lane1: digested product of T;
+
lane1: digested product of T;<br>
-
lane2: digested product of L;
+
lane2: digested product of L;<br>
-
lane3: marker;
+
lane3: marker;<br>
-
[[Image:PKU_20090730_Shuke_Wu_1.JPG|400px]]
+
[[Image:PKU_20090730_Shuke_Wu_1.JPG|400px]]<br>
-
The insert of T is about 900bp.  
+
The insert of T is about 900bp. <br>
The insert of L is about 1.4kb.  
The insert of L is about 1.4kb.  
-
'''DNA Gel purification:'''
+
'''DNA Gel purification:'''<br>
Inserts of L, T.   
Inserts of L, T.   
-
'''DNA ligation: '''
+
'''DNA ligation: '''<br>
{|cellpadding=5
{|cellpadding=5
|System||10uL
|System||10uL
Line 45: Line 58:
|T4 DNA ligase||1uL
|T4 DNA ligase||1uL
|}
|}
-
16℃ overnight.  
+
16℃ overnight. <br>
-
Insert: T *2;  
+
Insert: T *2; <br>
Vertor: tetP (has already digested by EcoR1 & Xba1)
Vertor: tetP (has already digested by EcoR1 & Xba1)
==='''2009.7.31'''===
==='''2009.7.31'''===
-
'''Transformation:'''  
+
'''Transformation:''' <br>
-
Products of ligation (T+tetP *2), competent cells 50uL each,  
+
Products of ligation (T+tetP *2), competent cells 50uL each, <br>
Smear to LB plate with Amp  
Smear to LB plate with Amp  
-
'''DNA ligation: '''
+
'''DNA ligation: '''<br>
{|cellpadding=5
{|cellpadding=5
|System||10uL
|System||10uL
Line 69: Line 82:
|T4 DNA ligase||1uL
|T4 DNA ligase||1uL
|}
|}
-
16℃ overnight.  
+
16℃ overnight. <br>
-
Insert: L *2;  
+
Insert: L *2; <br>
Vertor: lacP (has already digested by EcoR1 & Xba1, by He Siheng)
Vertor: lacP (has already digested by EcoR1 & Xba1, by He Siheng)
'''Transformation:'''  
'''Transformation:'''  
-
Products of ligation (L+lacP *2), competent cells 50uL each,  
+
Products of ligation (L+lacP *2), competent cells 50uL each, <br>
Smear to LB plate with Amp  
Smear to LB plate with Amp  
Line 81: Line 94:
Every plate is very well: more than 100 clones  
Every plate is very well: more than 100 clones  
-
'''PCR: (colony PCR, T-tetP)'''
+
'''PCR: (colony PCR, T-tetP)'''<br>
{|cellpadding=5
{|cellpadding=5
|System||10 uL
|System||10 uL
Line 89: Line 102:
|primer (standard primer)||0.5uL each
|primer (standard primer)||0.5uL each
|-
|-
-
|water||4uL template
+
|water||4uL
|-
|-
-
|10 colonies of T-tetP||
+
|template||10 colonies of T-tetP
|}
|}
   
   
-
'''Gel electrophoresis: (help by Lin Min)'''
+
'''Gel electrophoresis: (help by Lin Min)'''<br>
-
Refer to Lin Min’s notes,  
+
Refer to Lin Min’s notes, <br>
-
All of 10 colonies are wrong!!!
+
All of 10 colonies are wrong!!!<br>
Repeat!!!
Repeat!!!
-
'''Double digest: (again, tetP)'''
+
'''Double digest: (again, tetP)'''<br>
-
tetP:
+
tetP:<br>
{|cellpadding=5
{|cellpadding=5
|EcoR1||1uL
|EcoR1||1uL
Line 114: Line 127:
37 ℃ 4 hour
37 ℃ 4 hour
-
'''Transfer colonies: (L-lacP)'''
+
'''Transfer colonies: (L-lacP)'''<br>
Transfer 6 colonies (L-lacP) into 5ml LB, and amplify the Ecoli.  
Transfer 6 colonies (L-lacP) into 5ml LB, and amplify the Ecoli.  
-
'''Plasmid mini prep: (L-lacP)'''
+
'''Plasmid mini prep: (L-lacP)'''<br>
6 colonies of L-lacP
6 colonies of L-lacP
-
'''Double digest: (to check the correct L-lacP)'''
+
'''Double digest: (to check the correct L-lacP)'''<br>
-
6 L-lacP:
+
6 L-lacP:<br>
{|cellpadding=5
{|cellpadding=5
|EcoR1||1uL
|EcoR1||1uL
Line 137: Line 150:
==='''2009.8.2'''===   
==='''2009.8.2'''===   
-
'''Gel electrophoresis: (check the correct L-lacP)'''
+
'''Gel electrophoresis: (check the correct L-lacP)'''<br>
-
Products of double digest  
+
Products of double digest <br>
-
Marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb
+
Marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb<br>
-
loading buffer and DNA dye: 6×
+
loading buffer and DNA dye: 6×<br>
-
Voltage and time: 60V 5min; 120V 15min
+
Voltage and time: 60V 5min; 120V 15min<br>
-
[[Image:PKU_20090802_Shuke_Wu_1.JPG|400px]]
+
[[Image:PKU_20090802_Shuke_Wu_1.JPG|400px]]<br>
I forgot to add the Marker, but from the result we can easily find that all these 6 colonies are wrong!!!
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)'''
+
'''PCR: (colony PCR, L-lacP)'''<br>
{|cellpadding=5
{|cellpadding=5
|System||10 uL
|System||10 uL
Line 153: Line 166:
|primer (standard primer)||0.5uL each
|primer (standard primer)||0.5uL each
|-
|-
-
|water||4uL template
+
|water||4uL
|-
|-
-
|24 colonies of L-lacP||
+
|template||24 colonies of L-lacP
|}  
|}  
-
'''Gel electrophoresis: (check the correct L-lacP)'''
+
'''Gel electrophoresis: (check the correct L-lacP)'''<br>
-
Products of PCR  
+
Products of PCR <br>
-
Marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb
+
Marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb<br>
-
loading buffer and DNA dye: 6×
+
loading buffer and DNA dye: 6×<br>
-
Voltage and time: 60V 5min; 120V 15min
+
Voltage and time: 60V 5min; 120V 15min<br>
-
[[Image:PKU_20090802_Shuke_Wu_2.JPG|400px]]
+
[[Image:PKU_20090802_Shuke_Wu_2.JPG|400px]]<br>
-
From the lane 5 to the last one are 24 results of L-lacP PCR.  
+
From the lane 5 to the last one are 24 results of L-lacP PCR. <br>
-
Since there is not any DNA larger than 1kb, all of these 24 colonies are wrong!!!
+
Since there is not any DNA larger than 1kb, all of these 24 colonies are wrong!!!<br>
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!!!
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)'''
+
'''Double digest: (again, lacP)'''<br>
-
lacP:
+
lacP:<br>
{|cellpadding=5
{|cellpadding=5
|EcoR1||1uL
|EcoR1||1uL
Line 183: Line 196:
37 ℃ overnight!
37 ℃ overnight!
-
'''DNA ligation (again T+tetP): '''
+
'''DNA ligation (again T+tetP): '''<br>
{|cellpadding=5
{|cellpadding=5
|System||10uL
|System||10uL
Line 197: Line 210:
|T4 DNA ligase||1uL
|T4 DNA ligase||1uL
|}
|}
-
16℃ 4 hours  
+
16℃ 4 hours <br>
-
Insert: T *2;  
+
Insert: T *2; <br>
Vertor: tetP (digested on Aug.1st)
Vertor: tetP (digested on Aug.1st)
-
'''Transformation: (again T+tetP)'''
+
'''Transformation: (again T+tetP)'''<br>
-
Products of ligation (T+tetP *2), competent cells 50uL each,  
+
Products of ligation (T+tetP *2), competent cells 50uL each, <br>
Smear to LB plate with Amp  
Smear to LB plate with Amp  
==='''2009.8.3'''===
==='''2009.8.3'''===
-
'''PCR product purification:'''  
+
'''PCR product purification:''' <br>
lacP (digested yesterday)
lacP (digested yesterday)
-
'''DNA ligation (again L+lacP): '''
+
'''DNA ligation (again L+lacP): '''<br>
{|cellpadding=5
{|cellpadding=5
|System||10uL
|System||10uL
Line 224: Line 237:
|T4 DNA ligase||1uL
|T4 DNA ligase||1uL
|}
|}
-
16℃ 4 hours  
+
16℃ 4 hours <br>
-
Insert: L *2;  
+
Insert: L *2; <br>
Vertor: lacP (digested on Aug.2nd )
Vertor: lacP (digested on Aug.2nd )
-
'''PCR: (colony PCR, T-tetP)'''
+
'''PCR: (colony PCR, T-tetP)'''<br>
{|cellpadding=5
{|cellpadding=5
|System||10 uL
|System||10 uL
Line 236: Line 249:
|primer (standard primer)||0.5uL each
|primer (standard primer)||0.5uL each
|-
|-
-
|water||4uL template
+
|water||4uL
|-
|-
-
|10 colonies of T-tetP||
+
|template||10 colonies of T-tetP
|}
|}
-
'''Gel electrophoresis:'''  
+
'''Gel electrophoresis:''' <br>
-
Products of PCR  
+
Products of PCR <br>
-
Marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb
+
Marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb<br>
-
loading buffer and DNA dye: 6×
+
loading buffer and DNA dye: 6×<br>
-
Voltage and time: 60V 5min; 120V 15min
+
Voltage and time: 60V 5min; 120V 15min<br>
-
[[Image:PKU_20090803_Shuke_Wu_1.JPG|400px]]
+
[[Image:PKU_20090803_Shuke_Wu_1.JPG|400px]]<br>
-
Since there is not any DNA larger than 1kb, all of these 10 colonies are wrong Again!!!
+
Since there is not any DNA larger than 1kb, all of these 10 colonies are wrong Again!!!<br>
Bad Luck!!!!!
Bad Luck!!!!!
-
'''Transformation: (again L+lacP)'''
+
'''Transformation: (again L+lacP)'''<br>
-
Products of ligation (L+ lacP *2), competent cells 50uL each,  
+
Products of ligation (L+ lacP *2), competent cells 50uL each, <br>
Smear to LB plate with Amp  
Smear to LB plate with Amp  
-
'''Double digest: (the 3rd time! tetP and T)'''
+
'''Double digest: (the 3rd time! tetP and T)'''<br>
-
tetP:
+
tetP:<br>
{|cellpadding=5
{|cellpadding=5
|EcoR1||1uL
|EcoR1||1uL
Line 267: Line 280:
|water||12uL
|water||12uL
|}
|}
-
T:
+
T:<br>
{|cellpadding=5
{|cellpadding=5
|pe1||1uL
|pe1||1uL
Line 283: Line 296:
==='''2009.8.4'''===  
==='''2009.8.4'''===  
-
'''PCR product purification:'''  
+
'''PCR product purification:''' <br>
tetP (digested yesterday)
tetP (digested yesterday)
-
'''Gel electrophoresis:'''
+
'''Gel electrophoresis:'''<br>
-
Products of double digest of L, T  
+
Products of double digest of L, T <br>
-
marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb
+
marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb<br>
-
loading buffer and DNA dye: 6×
+
loading buffer and DNA dye: 6×<br>
-
voltage and time: 60V 5min; 120V 30min
+
voltage and time: 60V 5min; 120V 30min<br>
-
lane1: digested product of T;
+
lane1: digested product of T;<br>
-
lane2: marker
+
lane2: marker<br>
-
[[Image:PKU_20090804_Shuke_Wu_1.JPG|400px]]
+
[[Image:PKU_20090804_Shuke_Wu_1.JPG|400px]]<br>
The insert should be 900 bp, and it is correct!  
The insert should be 900 bp, and it is correct!  
-
'''DNA Gel purification:'''
+
'''DNA Gel purification:'''<br>
Insert of T.   
Insert of T.   
-
'''DNA ligation (the 3rd time T+tetP):'''  
+
'''DNA ligation (the 3rd time T+tetP):''' <br>
{|cellpadding=5
{|cellpadding=5
|System||10uL
|System||10uL
Line 313: Line 326:
|T4 DNA ligase||1uL
|T4 DNA ligase||1uL
|}
|}
-
16℃ 4 hours  
+
16℃ 4 hours <br>
-
Insert: T *2 (new);  
+
Insert: T *2 (new);<br>
Vertor: tetP (new);  
Vertor: tetP (new);  
-
'''Transformation: (the 3rd time T+tetP)'''
+
'''Transformation: (the 3rd time T+tetP)'''<br>
-
Products of ligation (T+tetP *2), competent cells 50uL each,  
+
Products of ligation (T+tetP *2), competent cells 50uL each, <br>
Smear to LB plate with Amp
Smear to LB plate with Amp
-
'''PCR: (the 2nd time colony PCR, L-lacP)'''
+
'''PCR: (the 2nd time colony PCR, L-lacP)'''<br>
{|cellpadding=5
{|cellpadding=5
|System||10 uL
|System||10 uL
Line 329: Line 342:
|primer (standard primer)||0.5uL each
|primer (standard primer)||0.5uL each
|-
|-
-
|water||4uL template
+
|water||4uL
|-
|-
-
|12 colonies of L-lacP||
+
|template||12 colonies of L-lacP
|}  
|}  
-
'''Gel electrophoresis: (check the correct L-lacP)'''
+
'''Gel electrophoresis: (check the correct L-lacP)'''<br>
-
Products of PCR  
+
Products of PCR <br>
-
Marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb
+
Marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb<br>
-
loading buffer and DNA dye: 6×
+
loading buffer and DNA dye: 6×<br>
-
Voltage and time: 60V 5min; 120V 15min
+
Voltage and time: 60V 5min; 120V 15min<br>
-
[[Image:PKU_20090804_Shuke_Wu_2.JPG|400px]]
+
[[Image:PKU_20090804_Shuke_Wu_2.JPG|400px]]<br>
-
The insert of correct L-lacP is about 1.4kb!!!
+
The insert of correct L-lacP is about 1.4kb!!!<br>
9 of 12 colonies are correct!!!!!
9 of 12 colonies are correct!!!!!
==='''2009.8.5'''===
==='''2009.8.5'''===
   
   
-
'''PCR: (the 3rd time colony PCR, T-tetP)'''
+
'''PCR: (the 3rd time colony PCR, T-tetP)'''<br>
{|cellpadding=5
{|cellpadding=5
|System||10 uL
|System||10 uL
Line 353: Line 366:
|primer (standard primer)||0.5uL each
|primer (standard primer)||0.5uL each
|-
|-
-
|water||4uL template
+
|water||4uL
|-
|-
-
|12 colonies of T-tetP||
+
|template||12 colonies of T-tetP
|}  
|}  
-
'''Gel electrophoresis: (check the correct T-tetP)'''
+
'''Gel electrophoresis: (check the correct T-tetP)'''<br>
-
Products of PCR  
+
Products of PCR <br>
-
Marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb
+
Marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb<br>
-
loading buffer and DNA dye: 6×
+
loading buffer and DNA dye: 6×<br>
-
Voltage and time: 60V 5min; 120V 60min
+
Voltage and time: 60V 5min; 120V 60min<br>
-
Lane 1~12: T-tetP 1~12
+
Lane 1~12: T-tetP 1~12<br>
-
Lane 13: Marker
+
Lane 13: Marker<br>
-
The insert is about 1kb, and 9 of these 12 colonies are CORRECT!!!!  
+
The insert is about 1kb, and 9 of these 12 colonies are CORRECT!!!! <br>
[[Image:PKU_20090805_Shuke_Wu_1.JPG|400px]]
[[Image:PKU_20090805_Shuke_Wu_1.JPG|400px]]
==='''Result'''===  
==='''Result'''===  
 +
At last, I successfully constructed: Pcat-2M-lacI/tetR-term+lacP/tetP, and they are the parts K228817/18.
At last, I successfully constructed: Pcat-2M-lacI/tetR-term+lacP/tetP, and they are the parts K228817/18.
==='''Experience'''===
==='''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.
 
-
 
-
 
-
 
-
 
 +
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 better digest it overnight. If you want to quick such as in two hours, reduce the amount of plasmid.
{{PKU_Beijing/Foot}}
{{PKU_Beijing/Foot}}
__NOTOC__
__NOTOC__

Latest revision as of 18:37, 21 October 2009
 
Notebook > AND Gate 1 > Input > Molecular cloning: Pcat-2M-lacI/tetR-term+lacP/tetP

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:

Spe11uL
Pst11uL
plasmid10uL
Buffer2uL
water6uL

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
template10 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
template24 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
template10 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
template12 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
template12 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 better digest it overnight. If you want to quick such as in two hours, reduce the amount of plasmid.

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