Team:Shanghai Metro Utd/Experiments

Shanghai_Metro_Utd

EXPERIMENTS

Experiment 1：Gradient PCR (20μl)

Protocol:

1. Mix the following reagents

2. PCR

Experiment 2：Bacterial colony PCR (20μl)

Protocol:

1. Isolating Single Colony from LB Plate Covered by Kanamycin

1) Mark and number the bacterial colony on the plate.

2) Pick a monoclonal population from the plate by a sterile pipette tip and place it in a test tube with LB 4 mL of antibiotic-free LB liquid medium.

3) Place the tube in a shaking incubator at 37℃ for 12 h.

2. PCR

Experiment 3：Gel Electrophoresis (2% Agarose)

Protocol:

1. Weigh 0.6g agarose.

2. Add 1960 mL distilled water to dilute 40ml 50X TAE buffer into 2L 1X TAE buffer solution.

3. Measure 30 mL 1X TAE buffer solution.

4. Mix agarose with prepared TAE buffer solution.

5. Heat in microwave for 3 min until the solution clarifies.

6. Cool down the solution at room temperature until hands can be comfortably kept on the flask.

7. Shake gently to mix well and pour the agarose solution into the gel box and wait for it to solidify.

Experiment 4：LB Culture Medium

1. Liquid 500 mL

2. Solid 200 mL

Experiment 5：Plasmid Extraction (Carrier plasmid PET28a)

Protocol:

1. Add 500μL of Buffer BL to the spin column. Centrifuge at 13,400×g for 1 min. Decant the filtrate in the collection tube.

2. Add 5-15ml overnight LB culture. Centrifuge at 13,400×g for 1 min. Collect the supernatant.

3. Add 500μl of Buffer P1. Resuspend the bacterial pellet. Besure that the bacteria pellet is completely resuspended.

4. Transfer to the microfuge tube. Add 500μl Buffer P2 and gently inverting the tube for 6-8 times until a clear solution is formed.

5. Add 500μl Buffer P4 and gently inverting the tube for 4-6 times and sit quietly for 10 min and centrifuge at 13,400×g for 10 min.

6. Add the supernatant to the filter tube CS and centrifuge at 13,400×g for 2 min.

7. Add 0.3× isopropanol to the filtrate and gently mixing and transfer to CP4 spin column.

8. Centrifuge at 13,400×g for 1 min and discard the filtrate.

9. Add protein PD and centrifuge at 13,400×g for 1 min and discard the filtrate.

10. Add 600 μl buffer PW and sit quietly for 2-5 min and centrifuge at 13,400×g for 1 min

11. Repeat step 10 and centrifuge at 13,400×g for 1 min

12. Place CP4 in a clean microfuge tube and add 30μL heated buffer TB to the absorption film and sit quietly for 2 min and centrifuge at 13,400×g for 1 min and collect the solution in the collection tube.

Experiment 6: Bacterial Transformation

Protocol:

1. Take out the competence from the fridge (-81℃) and put it in an ice box.

2. Due to the experimental requirement, we add 9μl TB to dilute 1μl 262.5ng/μl plasmid into 26.25ng/μl.

3. Add 1μl diluted plasmid into 50μl competence, and put it in the ice box for 30 minutes.

4. Transfer the sample to the 42℃ water bath for 90s.

5. Immediately iced the sample for 5 minutes.

6. Add 500μl LB solution to the sample and cultivate it at 37℃ for 40 minutes.

7. Centrifuge the sample at 800xg for 5 minutes.

8. Use an alcohol burner to sterilize glass rods and set the glass rods aside for 5~10 minutes.

9. Discard 450μl supernatant with a pipette.

10. Mix the the precipitate with the supernatant left in the tube.

11. Use a pipette to transfer the mixture onto a board with 10㎍/mL Kanamycin

12. Use the glass rod to spread the mixture evenly on the board and cultivate the mixture on the inverted board for 12~15hs.

Experiment 7: Restriction Enzyme Digestion

Protocol:

1. Cut vector

1) Mix the following reagents:

2) Centrifuge for a few seconds

2) Centrifuge for a few seconds 3) PCR for 1h under 37℃.

2. Cut targeted gene

1) Mix the following reagents:

Experiment 8: DNA Gel Extraction (Performed according to AxyPrep DNA Gel Extraction Kit)

1. Reagent:

2. Caution:

1) Cutting the DNA-containing gel into small pieces (DNA-containing gel can be seen under the UV light) to decrease the surface area and increase the extraction rate. Do not expose DNA-containing gel under the UV light for long periods of time to reduce UV damage to DNA.

2) The gel must be completely dissolved for better extraction rate.

3) The eluent should be pre-warmed to 65°C to increase the elution efficiency.

3. Preparation Before Experiment:

1) Before using, ethyl alcohol should be added to Buffer W2.

2) Make sure that the pipette tip and microfuge tube are without nucleic acid contamination and no precipitations are in Buffer.

3) Adjust the water bath to 65°C.

4. Protocol:

1) Excise the DNA-containing agarose gel slice with a clean, sharp scalpel under ultraviolet illumination. Briefly place the excised gel slice on absorbent toweling to remove redundant buffer. Mince the gel into small pieces, transfer to a 2.0 ml microfuge tube.

2) Add a 250 μL Buffer BL. Resuspend the gel in Buffer BL by vortexing and heating at 65°C. Intermittently vortexing (every 2-3 minutes) until the gel is completely dissolved (typically, 6-8 min later) and the color of the dissolved gel should be light yellow.

3) Transfer the solution to EC and centrifuge at 12,000 xg 22°C for 1 min and discard the collection tube.

4) Add 700 μL Buffer W2 to EC and centrifuge at 12000 xg 22°C for 1 min and discard the collection tube.

5) Repeat step 4.

6) Transfer EC to the empty collection tube and centrifuge at 12000 xg 22°C for 2 mins.

7) Transfer EC to the clean 1.5 ml microfuge tube and stew for 2 mins with lids opened and add 50 μL pre-warmed Eluent and stew for 2 mins, then centrifuge at 12000 xg 22°C for 2 mins.

8) Transfer the Eluent in the 1st microfuge tube to the 2nd microfuge tube and stew for 2 mins with lids opened and centrifuge at 12000 xg 22°C for 2 mins.

9) Repeat step 8 for until all solutions are eluted.

Experiment 9: SDS-PAGE (Performed according to Biochemistry Experiment and Guidance of China Pharmaceutical University)

1. Mix the following reagents: (TEMED is added last)

1) Monomer running gel

2) Monomer Stacking gel

2. Protocol:

1) After the glass plates are aligned, put them into the rubber frame and clamp them (the plates must be aligned to avoid leakage). Then clamp the clip.

2) Use pipette to extract 4 ml of running gel solution and release it into spaces between parallel glass, until it reaches 5 mm below the line. Create a flat surface above the running gel solution by adding a water layer on top of the solution. Allow the gel to polymerize for at least 30 min.

3) When there is a refracting line between running gel and water layer, pour off the water. Wash the gel for several times with deionized water and blot the water with filter paper.

4) Pour the stacking gel above the running gel.

5) Immediately insert the comb to create sample wells in the stacking gel before it polymerizes (keep the comb level). Be sure that the comb is clean and that no air bubbles form. Allow the gel to polymerize for at least 30 minutes.

Experiment 10: Protein Induction and Expression

Protocol:

1. Pick a single colony of the transformed BL21 in 4 ml LB (containing 100 μg/ml kanamycin), and culture it overnight at 37°C.

2. Inoculate the bacterial colony into 1L LB (containing 50 μg/ml kanamycin), and continue to cultivate it until the value of OD is 0.6 (with LB as the control).

3. Add IPTG to a concentration of 1 mmol/L.

4. Induce expression at 37°C for 3 h.

5. Centrifuged at 4°C at 4000 rpm for 10 min.

6. Collect bacterial pellets.

Experiment 11: Preparation and dissolution of inclusion bodies

Protocol:

1. Add 50 ml of buffer (50 mmol/l Tris-HCl, pH 8.0, 2 mmol/L EDTA) to the E. coli collected from centrifugation.

2. Vortex the bacterial suspension.

3. Fissure the bacteria with a High Pressure Homogenizer.

4. Centrifuge at 4℃ at 12000 rpm for 20 min and collect the precipitate.

5. Resuspend the inclusion bodies with 50 ml inclusion body washing solution (50 mmol/l Tris-HCl, pH 8.0, 2 mmol/L EDTA, 1% Triton X-100), and centrifuge at 4°C at 12000 rpm for 20 min.

6. Repeat step 5 and collect inclusion bodies.

7. Conduct 15% SDS electrophoresis to analyze the bacterial liquid before and after induction, inclusion bodies and supernatant after broken bacterial cells.

8. Add the above inclusion bodies to 20 ml of inclusion body denaturation solution (7 mol/L guanidine hydrochloride, 0.15 mol/L GSH, 0.1 mol/L Tris-HCl, pH 8.1, 2 mmol/L EDTA)

9. Stir and dissolve in an anaerobic environment for 2 hours.

10. Centrifuge at 4000r for 20 minutes to take the supernatant, which is the denatured protein solution.

Experiment 12: Protein Refolding

Protocol:

1. Slowly drop the denatured protein solution into 500 ml refolding buffer (0.5 mol/L L-Arginine, 0.6 mmol/L GSSG, pH 8.0) which has been filtered with 0.22 μm microporous filter before use.

2. After all the denaturation solution is added to the refolding buffer, continue to stir the refolding buffer for about 1 h.

3. Place the solution overnight at 4°C, and dilute to 2 L with ddH2O.

Experiment 13: Protein Purification

Protocol:

1. Pack SP Beads 6FF gel stored in 20% ethanol on the column (about 10 ml).

2. Equilibrate 3 column volumes with buffer A (10 mM Tris-HCl, pH 8.0, 0.2 M NaCl).

3. Load the refolded protein solution filtered by a 0.22 μm filter membrane and pass it through the column.

4. Analyze the solution before and after passing the column by SDS-PAGE.

5. Wash with buffer A for about one column volume and collect the washed solution.

6. Elute with buffer B (10 mM Tris-HCl, pH 8.0, 0.8 M NaCl) for more than 5 column volumes, and continuously collect the eluted solution for analysis by SDS-PAGE.

7. Concentrate the eluted protein solution with an ultrafiltration tube and replace it with ddH2O.

8. Freeze the protein solution into a solid with liquid nitrogen.

9. Lyophilized the protein solid with a freeze dryer to obtain a protein powder.

Experiment 14: Protein Activity Verification I - RNA Extraction

Protocol:

1. Resuspend the HEK293-GFP cell pellet with 2 ml Trizol to a 15 ml centrifuge tube.

2. Leave it at room temperature for 5 min.

3. Add 0.4 ml of chloroform and shake for 15 seconds.

4. Leave it at room temperature for 5 min

5. Centrifuge it at 12000 rpm at 4°C for 15 min. (At this time, the liquid is divided into three layers, the lower red phase, the middle organic phase, and the upper colorless phase.)

6. Carefully transfer the upper colorless phase to a clean 1.5ml Ep tube. (The volume of the upper colorless water sample layer is about 60% of the volume of Trizol added.)

7. Add an equal volume of pre-cooled isopropanol.

8. Leave it at room temperature for 10 minutes to precipitate RNA.

9. Centrifuge at 12000 rpm at 4°C for 10 minutes. (At this time, the sediment can be seen at the bottom of the tube. If the there is a large amount of sediment, you can see the white sediment in the form of flakes, and if there is less, you can see granular sediment.)

10. Carefully remove the supernatant.

11. Add 2 ml of 75% ethanol, blow with a gun several times.

12. Centrifuge at 4°C at 7500 rpm for 5 min.

13. Discard the supernatant and wait till the ethanol evaporates and dries.

14. Add 40μl of enzyme-free water to resuspend all RNA, and measure the RNA concentration and OD value with Nanodrop.

15. Store the RNA at -80℃.

Experiment 15: Protein Activity Verification II - RNA Degradation

Protocol:

1. Replace the electrophoresis solution in the electrophoresis tank with a new one.

2. Prepare 3 reaction systems with PCR tubes.

1) 50 μl: 0.33 mmol/L Hepes, 0.33 mol/L NaCl, 883.82 ng/μl total RNA, 10 μmol/L rANG;

2) 50 μl: 0.33 mmol/L Hepes, 0.33 mol/L NaCl, 883.82 ng/μl total RNA, 1 μmol/L rANG;

3) 50 μl: 0.33 mmol/L Hepes, 0.33 mol/L NaCl, 883.82 ng/μl total RNA, 0.2 mg/ml RNaseA.

3. Metal bath at 37°C for 1 h.

4. Take 50 μl and add 10 μl 6X gel loading to electrophoresis. (1% agarose gel electrophoresis, 140 V, 40 min)

Experiment 16: Interaction study between heparin and Angiogenin

Protocol:

1. Add 1mg/ml Heparan Sulfate to the transparent flat bottom 96-hole plate, cultivate for 2 hours.
2. Wash the plate with PBS 3 times, 5 minutes each time.
3. Add 3% concentration of BSA, react for 1 hour. Add 100ul angiogenin with different concentrations to each hole, react for 1 hour. Wash the plate with PBS for 3 times.
4. Add anti-His (1:2000) coupled with HRP, react for 1 hour. Wash the plate with PBS 6 times.
5. Add TMB, react for 30 minutes. Add 2 mol H2SO4.
6. Use SpectraMax M5e to measure the absorption value.