Team:Tsinghua/Notebook

From 2008.igem.org

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<!--- The Mission, Experiments --->
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{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="100%" align="center"
!align="center"|[[Team:Tsinghua|HOME]]
!align="center"|[[Team:Tsinghua|HOME]]
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='''Basic Wet-lab protocols'''=
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='''Basic Wet-Lab Protocols'''=
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|align="center"|HOLD
|align="center"|HOLD
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Incubate at 37℃, 1.5 hrs or longer (Enzymes from Takara Co.Ltd or NEB).
Incubate at 37℃, 1.5 hrs or longer (Enzymes from Takara Co.Ltd or NEB).
 +
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Incubate at 16-18℃,1hr or longer (Ligation Kit from Takara Co.Ltd).
Incubate at 16-18℃,1hr or longer (Ligation Kit from Takara Co.Ltd).
 +
 +
'''5. Transformation'''
'''5. Transformation'''
 +
 +
1. Place TOP10 cells (Transgen, 100uL per well) onto ice for 10 min.
 +
 +
2. Add 10uL ligation mixture to the cells.
 +
 +
3. Place the cells on ice for 20min.
 +
 +
4. Heat shock at 42℃ for 1min, then place on ice for 2min.
 +
 +
5. Add 900uL LB without antibiotics, and shake at 37℃ for 1h.
 +
 +
6. Centrifuge at 8,000rpm for 1min.
 +
 +
7. Decant the supernatant, resuspend the cell pellet in 200uL LB, and spread the cells on LB plates of corresponding antibiotics.
 +
 +
 +
 +
 +
 +
='''Self-modified Protocols'''=
 +
 +
'''1. Advanced Protocol for Parts Extraction'''
 +
 +
We have revised the Parts Extraction Protocol and obtained a higher efficiency of transformation. Here are some details:
 +
 +
1. Dissolve the plasmid in Elution Buffer for 30min or longer to get a higher concentration of plasmids.
 +
 +
2. Shake cells at 37℃ for 2h or longer before spread. This would help the cells recover from heat shock.
 +
 +
''By Yilong Zou''
 +
-
='''Wet-lab Notes'''=
 
-
'''1. Advanced protocol for parts extraction'''
 
'''2. BioBrick Parts Making Protocol'''
'''2. BioBrick Parts Making Protocol'''
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ELSE  
ELSE  
-
    GOTO STEP9
+
GOTO STEP9
-
2. Design primers with half-prefix (Xba1) and half-suffix (Spe1)
+
2. Design primers with half-prefix (Xba1) and half-suffix (Spe1)
-
3. PCR from according genome/plasmid
+
3. PCR from according genome/plasmid
-
4. Purify PCR product using Gel Extraction Kit (Transgen)
+
4. Purify PCR product using Gel Extraction Kit (Transgen)
-
5. Digest with Xba1+Spe1
+
5. Digest with Xba1+Spe1 (Takara)
-
6. Ligation with pSB1AC3, which was digest with Xba1+Spe1 and treated with CIAP
+
6. Ligation with pSB1AC3, which was digest with Xba1+Spe1 (Takara) and treated with CIAP
-
7. Transform to TOP10 cells
+
7. Transform to TOP10 cells
-
8. Identify clones with colony PCR
+
8. Identify clones with colony PCR
-
    GOTO STEP20
+
GOTO STEP20
-
9. Design primers with full-prefix and full-suffix
+
9. Design primers with full-prefix and full-suffix
10. PCR from according genome/plasmid
10. PCR from according genome/plasmid
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11. Purify PCR product using Gel Extraction Kit (Transgen)
11. Purify PCR product using Gel Extraction Kit (Transgen)
-
    FOR EcoR1
+
FOR EcoR1
-
    GOTO STEP12
+
GOTO STEP12
-
    ELSE  
+
ELSE  
-
    GOTO STEP14
+
GOTO STEP14
-
12. Digest with Xba1+Pst1
+
12. Digest with Xba1+Pst1 (Takara)
-
13. Ligation with pSB1AC3, which was digest with Xba1+Pst1 and treated with CIAP
+
13. Ligation with pSB1AC3, which was digest with Xba1+Pst1 (Takara) and treated with CIAP
-
    GOTO STEP16
+
GOTO STEP16
-
14. Digest with EcoR1+Spe1
+
14. Digest with EcoR1+Spe1 (Takara)
-
15. Ligation with pSB1AC3, which was digest with EcoR1+Spe1 and treated with CIAP
+
15. Ligation with pSB1AC3, which was digest with EcoR1+Spe1 (Takara) and treated with CIAP
16. Transform to TOP10 cells
16. Transform to TOP10 cells
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20. Extract plasmid and send sequencing
20. Extract plasmid and send sequencing
-
    End^^
+
END ^^
 +
 
 +
''By Qi Liu''
 +
 
 +
 
 +
 
 +
 
 +
'''3. Quick & Easy Knock-out Protocol'''
 +
 
 +
(Reference 1)
 +
 
 +
1. Transform BW25113 with pKD46, incubate at 30℃ on LB plate with Amp+100.
 +
 
 +
2. Incubate BW25113-pKD46 in 100mL LB (Amp+100) at 30℃ until OD600=0.5~0.1. Add 1mL L-arabinose and incubate until OD600=0.6.
 +
 
 +
3. Place the cells on ice for 1h, wash with cooled water twice and 10% glycerol once, resuspend with 100uL 10% glycerol to make the competent cells of BW25113-pKD46.
 +
 
 +
4. Transform BW25113-pKD46 with 10uL PCR product of the DNA segment (for short, S) to be knock-out(30ng/uL), incubate at 30℃ on LB plate with Kan+50.
 +
 
 +
5. Identify clones using clony PCR with S-upstream + Kan, S-downstream + Kan, and S-upstream and S-downstream.
 +
 
 +
6. Incubate at 37℃ without antibiotics to throw out pKD46.
 +
 
 +
7. Select clones with Amp+100 (the cells would die) and Kan+50 (the cells would survive) LB plates.
 +
 
 +
8. Treat the selected cells as described in Step 2&3, then transform with pCP20, and incubate at 30℃ on LB plate with Amp+100.
 +
 
 +
9. Pick out several clones and incubate them at 42℃ for 24h.
 +
 
 +
10. Select clones with Amp+100 and Kan+50 LB plates(the cells would die on both plate).
 +
 
 +
11. Further identify clones using clony PCR with S-upstream and S-downstream.
 +
 
 +
12. Extract plasmid and send sequencing.
 +
 
 +
''By Yicheng Long''
 +
 
 +
 
 +
 
 +
'''Reference'''
 +
 
 +
1. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. PNAS. 2000, 97(12):6640-6645.
 +
 
 +
 
='''Wet-Lab Procedures'''=
='''Wet-Lab Procedures'''=
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{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="100%" align="center"
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!align="center"|[[Team:Tsinghua|HOME]]
 +
!align="center"|[[Team:Tsinghua/Team|Team]]
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!align="center"|[[Team:Tsinghua/Project|Project 1]]
 +
!align="center"|[[Team:Tsinghua/Project2|Project 2]]
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!align="center"|[[Team:Tsinghua/Parts|Parts]]
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!align="center"|[[Team:Tsinghua/Modelling|Modelling]]
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!align="center"|[[Team:Tsinghua/Notebook|Notebook]]
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!align="center"|[[Team:Tsinghua/Doodle|Doodle Board]]
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<!--- The Mission, Experiments --->
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[[image:06.jpeg|center|]]

Latest revision as of 04:31, 30 October 2008

331.jpeg
HOME Team Project 1 Project 2 Parts Modelling Notebook Doodle Board



Basic Wet-Lab Protocols

1. PCR

PCR System
Reagent Concentration/Activity Volume (50uL System) Volume (100uL System)
10x Pyrobest buffer II 10x 5 10
Pyrobest 0.3 0.5
dNTPmix 10mM each 1 2
Primer 1 10uM 1 2
Primer 2 10um 1 2
Template DNA changeable 0.5 1
MgCl2(Deletable) 0.2M 0.5 1
ddH2O 40.5 81

(Pyrobest DNA polymerase from Takara Co.Ltd.)


PCR Program
Step Condition Time
1 95℃ 5min
2 95℃ 30sec
3 [Tm(fu)-4]℃ 30sec
4 72℃ DNA length/kb/min
5 RETURN TO STEP 2 30-35 cycles
6 72℃ 10min
7 4℃ HOLD



2. Fusion PCR


The basic system is similar to common PCR. There are some notes to raise the fusion efficiency:

a. Complementary region length: 15-20bp

b. Raise the annealing temperature in the fusion step.


Fusion PCR Program
Step Condition Time
1 95℃ 5min
2 95℃ 30-50sec
3 {Tm(fu)+[(-2)~5]}℃ 40-80sec
4 72℃ DNA length/kb/min
5 RETURN TO STEP 2 10-15 cycles
6 72℃ 5min
7 Add amplification Primers
8 95℃ 2-5min
9 95℃ 30sec
10 [Tm(fu)-4]℃ 30sec
11 72℃ DNA length/kb/min
12 RETURN TO STEP 2 25-30 cycles
13 72℃ 10min
14 4℃ HOLD


3. Restriction Digestion

Restriction Digestion System
Reagent Concentration/Activity Volume(50uL system)
DNA <1ug
Restriction Enzyme buffer 10x 5uL
Enzyme 1 1uL
Enzyme 2 1uL
ddH2O to 50uL

Incubate at 37℃, 1.5 hrs or longer (Enzymes from Takara Co.Ltd or NEB).


4. Ligation

Ligation System
Reagent Volume(10uL system)
Solution I 5uL
DNA fragment 3.5uL(changeable)
Vector 1.5uL(changeable)

Incubate at 16-18℃,1hr or longer (Ligation Kit from Takara Co.Ltd).


5. Transformation

1. Place TOP10 cells (Transgen, 100uL per well) onto ice for 10 min.

2. Add 10uL ligation mixture to the cells.

3. Place the cells on ice for 20min.

4. Heat shock at 42℃ for 1min, then place on ice for 2min.

5. Add 900uL LB without antibiotics, and shake at 37℃ for 1h.

6. Centrifuge at 8,000rpm for 1min.

7. Decant the supernatant, resuspend the cell pellet in 200uL LB, and spread the cells on LB plates of corresponding antibiotics.



Self-modified Protocols

1. Advanced Protocol for Parts Extraction

We have revised the Parts Extraction Protocol and obtained a higher efficiency of transformation. Here are some details:

1. Dissolve the plasmid in Elution Buffer for 30min or longer to get a higher concentration of plasmids.

2. Shake cells at 37℃ for 2h or longer before spread. This would help the cells recover from heat shock.

By Yilong Zou



2. BioBrick Parts Making Protocol

1. get desired sequences through NCBI or other sources and check for restriction sites

FOR no (Xba1 or Spe1)

GOTO STEP2

ELSE

GOTO STEP9

2. Design primers with half-prefix (Xba1) and half-suffix (Spe1)

3. PCR from according genome/plasmid

4. Purify PCR product using Gel Extraction Kit (Transgen)

5. Digest with Xba1+Spe1 (Takara)

6. Ligation with pSB1AC3, which was digest with Xba1+Spe1 (Takara) and treated with CIAP

7. Transform to TOP10 cells

8. Identify clones with colony PCR

GOTO STEP20

9. Design primers with full-prefix and full-suffix

10. PCR from according genome/plasmid

11. Purify PCR product using Gel Extraction Kit (Transgen)

FOR EcoR1

GOTO STEP12

ELSE

GOTO STEP14

12. Digest with Xba1+Pst1 (Takara)

13. Ligation with pSB1AC3, which was digest with Xba1+Pst1 (Takara) and treated with CIAP

GOTO STEP16

14. Digest with EcoR1+Spe1 (Takara)

15. Ligation with pSB1AC3, which was digest with EcoR1+Spe1 (Takara) and treated with CIAP

16. Transform to TOP10 cells

17. Identify clones with colony PCR

18. Extract plasmid and site-directed mutate by fusion PCR

19. Transform to TOP10 cells

20. Extract plasmid and send sequencing

END ^^

By Qi Liu



3. Quick & Easy Knock-out Protocol

(Reference 1)

1. Transform BW25113 with pKD46, incubate at 30℃ on LB plate with Amp+100.

2. Incubate BW25113-pKD46 in 100mL LB (Amp+100) at 30℃ until OD600=0.5~0.1. Add 1mL L-arabinose and incubate until OD600=0.6.

3. Place the cells on ice for 1h, wash with cooled water twice and 10% glycerol once, resuspend with 100uL 10% glycerol to make the competent cells of BW25113-pKD46.

4. Transform BW25113-pKD46 with 10uL PCR product of the DNA segment (for short, S) to be knock-out(30ng/uL), incubate at 30℃ on LB plate with Kan+50.

5. Identify clones using clony PCR with S-upstream + Kan, S-downstream + Kan, and S-upstream and S-downstream.

6. Incubate at 37℃ without antibiotics to throw out pKD46.

7. Select clones with Amp+100 (the cells would die) and Kan+50 (the cells would survive) LB plates.

8. Treat the selected cells as described in Step 2&3, then transform with pCP20, and incubate at 30℃ on LB plate with Amp+100.

9. Pick out several clones and incubate them at 42℃ for 24h.

10. Select clones with Amp+100 and Kan+50 LB plates(the cells would die on both plate).

11. Further identify clones using clony PCR with S-upstream and S-downstream.

12. Extract plasmid and send sequencing.

By Yicheng Long


Reference

1. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. PNAS. 2000, 97(12):6640-6645.


Wet-Lab Procedures

  • Click on any day below to see what wet-lab procedures were conducted.
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