Aim

Activate E. coli DH5a/pSB1A3-mRFP, E. coli DH5a/pMD18T-insert

Preparation

1. Prepare medium and required materials

2. Clean bench, materials sterilization

3. Prepare diluted primer

4. Prepare configuration for amp mother solution (100mg/mL)

Experiment procedures

1. Spraying alcohol after wearing gloves before entering clean bench. Ignite alcohol lamp after drying gloves.

2. For sterilization, when we open/close the test tubes or bottles, we have to heat the opening of the test tubes or bottles with the alcohol lamp.

3. Using pipette, adding 5 mL LB medium.

4. Using pipette, adding 2.5μL Amp antibiotics

5. Using pipette, adding E. coli DH5a/pSB1A3-mRFP（or E. coli DH5a/pMD18T-insert）100μL

Aim

Extracte plasmids

Amplify the DNA using PCR

Double enzyme digestion

Agarose gel electrophoresis

Gel extraction

Experiment procedures

1. Extract the plamid

a. we use the alkaline lysis method to break the E. coli cell the extract 4 tubes of pSB1A3-mRFP and 1 tube of pMD18T-insert plasmids. This includes using the centrifuging machine to collect the pure DNA instead of the other impurities and carbohydrate that is decanting and washing repeatedly after centrifuging.

c.

d.Our result is reassured by examining dsDNA concentration and sample purity using Nanodrop.

e.

2. Proceeding PCR amplification program

3. Mix a. PrimeSTAR®️HS b. buffer c. primer 1 d. primer 2 e. template f. dNTP mixture g. sterile water to form a mixture

4. Mix 5μL mixture with 1μL 6xPS buffer

5. Proceeding electrophoresis by putting the mixture and loading marker into agarose gel

6. After electrophoresis, observing the result displayed on gel imaging system

7. Cut the gel then put into centrifuge tube

8. Recycle the gel

Aim

1. Prepare competent cells

2. Ligation

3. Transformation of linking products

Preparation

Precooling solutions: a. 0.1M CaCl2 b. 15% 0.1M CaCl2 in 4℃ fridge Experiment procedures

Experimrnt procedures

1. Ligation

2. Mix 1μL T4 DNA ligase, 6μL insert DNA, 1μL linear plasmid, 1μL 10 x DNA ligase buffer

3. Put the mixture in to PCR machine, at 16℃ for an hour

4. Prepare competent cells

5. Separate the E. coli solution, pour the LB meidum

6. Equally seprate the E. coli into two centrifuge tubes, then add 1 mL CaCl2 for each tube, mix the tube

7. Ice-bath fot 30 min

8. Centrifuge, pour the waste

9. Resuspend the E. coli with CaCl2

10. Add 1μL E.coli + CaCl2 solution to each centrifuge tube which contain ligation system

11. Ice-bath for 20 min

12. Water-bath at 42℃ for 1 min

13. Ice-bath for 10 min

14. Use glass spreading rod to apply on the medium evenly, then

15. seal

Results

Aim

Obtain positive cloning result

Preparation

1. sterile water 240μL

2. DNTP mix 24μL

3. Buffer 3μL

4. Primer 1 6μL

5. Primer 2 6μL

Experiment procedures

1. Taq mix: mix all the solutions from prepartions

2. Sperate the mixture into several centrifuge tubes

3. Mix 49μL taq mix and 1μL polymerase, then pick template DNA in the centrifuge tube

4. Add 0.6g agarose to 1Xbuffer, then heat

5. Add gene green into agarose solution, dyeing the gene

6. Use PCR system for electrophoresis

7. Select valid target gene fragments

8. Add ammonia benzyl to LB meidum

9. Add 5-10 Ml meidum with antibiotoic into the test tubes

10. Add the same template DNA into the test tubess

11. Sterlise the test tubes

12. Put test tubes into shaker

Aim

Extract the plasmid from E.coli prepared.

Tranfer the target gene to E.coli BL 21

Look under fluorescence microscope and examine the results

Experiment procedures

1. Extract plasmid from bacterial solution validated for the correct DHSa/ PSB1A3-target fragment

2. Transform the pSB1A3-fragment into E. coli BL21

3. Validation experiments by fluorescence microscopy

Results

  Through observation under fluorescence microscope, we found that the fluorescent protein was successfully expressed, which reveals the experiment was successful.