Team:SUSTC-Shenzhen-B/protocol

Lab Protocol

brief process

1. Site-Directed Mutagenesis

2. Restriction enzyme digestion and electrophoresis (proof)

3. Media Preparation

4. Bacterial Transformation

5. Colony PCR for verification

6. Culture the bacteria

7. Plasmid DNA Isolation

8. Restriction Enzyme Digestion and Electrophoresis(proof)

9. Amplify GFP & RFP

10. Electrophoresis and Restriction Enzyme Digestion.

11. Ligation

12. Bacterial Transformation

13. bacterial colony PCR for verification

14. Culture the bacteria

15. Plasmid DNA Isolation

16. Restriction Enzyme Digestion and Electrophoresis

Site-Directed Mutagenesis

We choose plasmid psb1a3 to be the vector that ligate GPF and RFP fragments.To protect the structural integrity of the constructed plasmid, we need to mutate a restriction enzyme cutting site named Pst I to Afl II. Proper primer are designed for this purpose.

PtoA-F:5'-CCACCTGACGTCTAAGAAAC-3'

PtoA-R:5'-ATGATCATCGCCGGCGAATTCAGGC-3'

1. Prepare the sample reaction as indicated below:

Total: 25μl

+ 0.25 μl of Ex Taq polymerase(酶的公司名称)

+ 2.5 μl of 10× Taq reaction buffer

+ 2.0 μl of dNTP(2mM)

+ 1.0 μl of template

+ 1.0 μl of oligonucleotide primer P1

+ 1.0 μl of oligonucleotide primer P2

+ 18.25 μl of ddH2O

2. Set thermocycler temperatures and the time. Procedure on the thermocycler are listed below:

① 94˚C for 5 min

② 30 cycle

a. 94˚C for 1 min

b. 55˚C for 1 min

c. 72˚C for 1 min20sec

③ 72˚C for 10 min

④ 4˚C for stock

Restriction Enzyme Digestion for Verification

After the PCR mutation we have to do a restriction enzyme digestion to test whether the mutation is successful. The reaction system is as follows:

1. Prepare the control reaction as indicated below:

Total: 10μl

+ 0.5μl of Pst I restriction enzyme

+ 1μl of 10XH buffer

+ 1μl of plasmid DNA

+ 7.5μl of ddH2O

2. Prepare the sample reaction as indicated below:

Total: 25μl

+ 0.5μl of Afl II restriction enzyme, #ER0831

+ 1μl of 10XM buffer

+ 1μl of plasmid DNA

+ 1.0μl of 0.1% BSA

+ 6.5μl of ddH2O

After the reaction, do an electrophoresis to see the 2k bp cyclic plasmid DNA change into linear. The process is as follows:

3. Prepare electrophoresis gel by adding 0.6g agarose to 60ml TAE (1% solution,1X,diluted from 50X TAE). Pour on conical flask and cover the Conical flask sealing surface with silver paper to avoid the loss of water vapor. Place in the microwave and microwave on middle for 1 minute at a time, pulling it out and swirling until solution is homogeneous again, then repeating (BE CAREFUL to watch the solution closely when swirling–it superheats and can boil over and cause severe burns). Continue until solution is seen clear and homogeneous with no exsistence of solid.Add 3 μl of (神马情况！！！)

4. By inserting the pipette tip below the TAE liquid and into the well, add 5μl of 1kb DNA ladder solution to first (and last if desired) well, skip one well, then begin adding the 5μl of digested DNA solutions mixed with 1μl loading buffer (6X) to the wells.

5. Place the cover on the electrophoresis unit, plug into the power source, and turn on voltage to 120V, set time to 30 minutes, and press the start button twice,leave when bubbles are seen. DNA separation can be observed as time goes on by turning off the power supply then gently removing the basin from the electrophoresis unit (be careful not to let the gel slip out of the basin) and placing on the UV transilluminator to see DNA bands.

6. When the desired level of separation is obtained, the basin can be placed on the transilluminator for picture taking(Of the absence of transilluminator,we use camera to take pictures with the UV light ).

Media Preparation

For all experiments involving the bacterial biomass and experimentation, proper media is chosen to grow the cells. Commonly,we use Lysogeny broth media for E. coli. The following is the media compositions and their quantities.

Lysogeny Broth (LB) liquid media (1 L)

Measure out these following:

Bacto-Tryptone - 10 g

NaCl - 10 g

Yeast Extract - 5 g

Add ddH2O and 5mmol/L Tris Buffer in a measuring cylinder to ensure accuracy, to make a total of 1 liter and pH is 8.0.

Lysogeny Broth (LB) solid media (1 L)

Measure out these following:

Bacto-Tryptone - 10 g

NaCl - 10 g

Yeast Extract - 5 g

Difco Agar - 15g

Add ddH2O and 5mmol/L Tris Buffer in a measuring cylinder to ensure accuracy, to make a total of 1 liter and pH is 8.0.

Autoclaving

Autoclave at 121 °C for 45 minutes (liquid setting, 0 minutes drying time). For making plates, after the media cool enough, antibiotics Ampicillin(0.6*10^-3 volume of media ) are added. At last media are poured 15ml on each plate and become solid.

Bacterial Transformation

Introduction of exogenous DNA into cells using non-viral methods is called “Transformation”.Transformation is commonly used to introduce recombinant plasmid DNA into bacterial strains which can transform naturally or can be made competitive for transformation by artificial means.

Depending on the expected transformation efficiency, there are two main types of competent cells that can be used for transformation.

1.Chemically competent cells

Chemically induced competent cells are calcium chloride-treated to facilitate attachment of the plasmid DNA to the competent cell membrane. During chemical transformation, the cells are heat-shocked in a water bath; which opens the pores of the cell membrane allowing entry of plasmid DNA from the buffer.

2.Electrocompetent cells

Electrocompetent cells are prepared for transformation using electroporation, a method that uses an electrical pulse to create pores through which genetic material enters the cells. This method usually has high transformation efficiency.