iGEM 2008 — Team Tsinghua

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# 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.

# **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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