Team:Tsinghua/Notebook

From 2008.igem.org

(Difference between revisions)
(→Notes)
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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
 +
6. Ligation with pSB1AC3, which was digest with Xba1+Spe1 and treated with CIAP
6. Ligation with pSB1AC3, which was digest with Xba1+Spe1 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
 +
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
 +
13. Ligation with pSB1AC3, which was digest with Xba1+Pst1 and treated with CIAP
13. Ligation with pSB1AC3, which was digest with Xba1+Pst1 and treated with CIAP
 +
Goto STEP16
Goto STEP16
 +
14. Digest with EcoR1+Spe1
14. Digest with EcoR1+Spe1
 +
15. Ligation with pSB1AC3, which was digest with EcoR1+Spe1 and treated with CIAP
15. Ligation with pSB1AC3, which was digest with EcoR1+Spe1 and treated with CIAP
 +
16. Transform to TOP10 cells
16. Transform to TOP10 cells
 +
17. Identify clones with colony PCR
17. Identify clones with colony PCR
 +
18. Extract plasmid and site-directed mutate by fusion PCR
18. Extract plasmid and site-directed mutate by fusion PCR
 +
19. Transform to TOP10 cells
19. Transform to TOP10 cells
 +
20. Extract plasmid and send sequencing
20. Extract plasmid and send sequencing
-
End^^67
+
 
 +
End^^LiuQi

Revision as of 11:54, 29 October 2008

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


Wet-lab Notes

Advanced protocol for parts extraction

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

6. Ligation with pSB1AC3, which was digest with Xba1+Spe1 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

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

Goto STEP16

14. Digest with EcoR1+Spe1

15. Ligation with pSB1AC3, which was digest with EcoR1+Spe1 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^^LiuQi


Wet-Lab Procedures

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