Team:Tsinghua/Notebook

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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.



Wet-lab Notes

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.

2. Shake cells at 37℃ for 2h or longer before spread



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

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 ^^

Wet-Lab Procedures

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