Difference between revisions of "Team:Shanghai Metro Utd/Experiments"

Latest revision as of 18:50, 19 October 2021

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.

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-<h1>Experiments</h1>
-<p>Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.</p>
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-If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the <a href="http://parts.igem.org/Main_Page">Registry</a>.
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-<h3>What should this page contain?</h3>
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-<li> Protocols </li>
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-<h3>Inspiration</h3>
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-<li><a href="https://2019.igem.org/Team:Nantes/Experiments">2019 Nantes </a></li>
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+    <div class="sub-content">
+        <div class="sub-title">EXPERIMENTS</div>
+        <div class="article-title black-font">Experiment 1：Gradient PCR (20μl)</div>
+        <div class="article-content">Protocol:</div>
+        <div class="article-content">1. Mix the following reagents</div>
+        <div class="img-wrap no-margin">
+            <img src="https://static.igem.org/mediawiki/2021/8/84/T--Shanghai_Metro_Utd--experiments01.jpg" alt="">
+        </div>
+        <div class="article-content">2. PCR</div>
+        <div class="img-wrap no-margin">
+            <img src="https://static.igem.org/mediawiki/2021/7/7d/T--Shanghai_Metro_Utd--experiments02.jpg" alt="">
+        </div>
+        <div class="article-title black-font">Experiment 2：Bacterial colony PCR (20μl)</div>
+        <div class="article-content">Protocol:</div>
+        <div class="article-content">1. Isolating Single Colony from LB Plate Covered by Kanamycin</div>
+        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;1) Mark and number the bacterial colony on the plate.</div>
+        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;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.</div>
+        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;3) Place the tube in a shaking incubator at 37℃ for 12 h.
+        </div>
+        <div class="article-content">2. PCR</div>
+        <div class="img-wrap no-margin">
+            <img src="https://static.igem.org/mediawiki/2021/c/c4/T--Shanghai_Metro_Utd--experiments03.jpg" alt="">
+        </div>
+        <div class="article-title black-font">Experiment 3：Gel Electrophoresis (2% Agarose)</div>
+        <div class="article-content">Protocol:</div>
+        <div class="article-content">1. Weigh 0.6g agarose.</div>
+        <div class="article-content">2. Add 1960 mL distilled water to dilute 40ml 50X TAE buffer into 2L 1X TAE buffer
+            solution.</div>
+        <div class="article-content">3. Measure 30 mL 1X TAE buffer solution.</div>
+        <div class="article-content">4. Mix agarose with prepared TAE buffer solution.</div>
+        <div class="article-content">5. Heat in microwave for 3 min until the solution clarifies.</div>
+        <div class="article-content">6. Cool down the solution at room temperature until hands can be comfortably kept
+            on the flask.</div>
+        <div class="article-content">7. Shake gently to mix well and pour the agarose solution into the gel box and
+            wait for it to solidify.</div>
+        <div class="article-title black-font">Experiment 4：LB Culture Medium</div>
+        <div class="article-content">1. Liquid 500 mL</div>
+        <div class="img-wrap no-margin">
+            <img src="https://static.igem.org/mediawiki/2021/c/ce/T--Shanghai_Metro_Utd--experiments04.jpg" alt="">
+        </div>
+        <div class="article-content">2. Solid 200 mL</div>
+        <div class="img-wrap no-margin">
+            <img src="https://static.igem.org/mediawiki/2021/a/af/T--Shanghai_Metro_Utd--experiments05.jpg" alt="">
+        </div>
+        <div class="article-title black-font">Experiment 5：Plasmid Extraction (Carrier plasmid PET28a) </div>
+        <div class="img-wrap no-margin">
+            <img src="https://static.igem.org/mediawiki/2021/6/6e/T--Shanghai_Metro_Utd--experiments06.jpg" alt="">
+        </div>
+        <div class="article-content">Protocol:</div>
+        <div class="article-content">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.</div>
+        <div class="article-content">2. Add 5-15ml overnight LB culture. Centrifuge at 13,400×g for 1 min. Collect the
+            supernatant.</div>
+        <div class="article-content">3. Add 500μl of Buffer P1. Resuspend the bacterial pellet. Besure that the bacteria
+            pellet is completely resuspended.</div>
+        <div class="article-content">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.</div>
+        <div class="article-content">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.</div>
+        <div class="article-content">6. Add the supernatant to the filter tube CS and centrifuge at 13,400×g for 2 min.
+        </div>
+        <div class="article-content">7. Add 0.3× isopropanol to the filtrate and gently mixing and transfer to CP4 spin
+            column.</div>
+        <div class="article-content">8. Centrifuge at 13,400×g for 1 min and discard the filtrate.</div>
+        <div class="article-content">9. Add protein PD and centrifuge at 13,400×g for 1 min and discard the filtrate.
+        </div>
+        <div class="article-content">10. Add 600 μl buffer PW and sit quietly for 2-5 min and centrifuge at 13,400×g for
+min</div>
+        <div class="article-content">11. Repeat step 10 and centrifuge at 13,400×g for 1 min</div>
+        <div class="article-content">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.</div>
+        <div class="article-title black-font">Experiment 6: Bacterial Transformation</div>
+        <div class="article-content">Protocol:</div>
+        <div class="article-content">1. Take out the competence from the fridge (-81℃) and put it in an ice box.</div>
+        <div class="article-content">2. Due to the experimental requirement, we add 9μl TB to dilute 1μl 262.5ng/μl
+            plasmid into 26.25ng/μl.</div>
+        <div class="article-content">3. Add 1μl diluted plasmid into 50μl competence, and put it in the ice box for 30
+            minutes.</div>
+        <div class="article-content">4. Transfer the sample to the 42℃ water bath for 90s.</div>
+        <div class="article-content">5. Immediately iced the sample for 5 minutes.</div>
+        <div class="article-content">6. Add 500μl LB solution to the sample and cultivate it at 37℃ for 40 minutes.
+        </div>
+        <div class="article-content">7. Centrifuge the sample at 800xg for 5 minutes.</div>
+        <div class="article-content">8. Use an alcohol burner to sterilize glass rods and set the glass rods aside for
+~10 minutes.</div>
+        <div class="article-content">9. Discard 450μl supernatant with a pipette.</div>
+        <div class="article-content">10. Mix the the precipitate with the supernatant left in the tube.</div>
+        <div class="article-content">11. Use a pipette to transfer the mixture onto a board with 10㎍/mL Kanamycin</div>
+        <div class="article-content">12. Use the glass rod to spread the mixture evenly on the board and cultivate the
+            mixture on the inverted board for 12~15hs.</div>
+        <div class="article-title black-font">Experiment 7: Restriction Enzyme Digestion </div>
+        <div class="article-content">Protocol:</div>
+        <div class="article-content">1. Cut vector</div>
+        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1) Mix the following reagents:
+        </div>
+        <div class="img-wrap no-margin">
+            <img src="https://static.igem.org/mediawiki/2021/c/ce/T--Shanghai_Metro_Utd--experiments07.jpg" alt="">
+        </div>
+        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2) Centrifuge for a few seconds
+        </div>
+        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2) Centrifuge for a few seconds
+) PCR for 1h under 37℃.
+        </div>
+        <div class="article-content">2. Cut targeted gene</div>
+        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1) Mix the following reagents:
+        </div>
+        <div class="img-wrap no-margin">
+            <img src="https://static.igem.org/mediawiki/2021/8/8b/T--Shanghai_Metro_Utd--experiments08.jpg" alt="">
+        </div>
+        <div class="article-title black-font" style="line-height: 32px;">Experiment 8: DNA Gel Extraction (Performed
+            according to AxyPrep DNA Gel
+            Extraction Kit)</div>
+        <div class="article-content">1. Reagent:</div>
+        <div class="img-wrap no-margin">
+            <img src="https://static.igem.org/mediawiki/2021/e/ee/T--Shanghai_Metro_Utd--experiments09.jpg" alt="">
+        </div>
+        <div class="article-content">2. Caution:</div>
+        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;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.</div>
+        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2) The gel must be completely
+            dissolved for better extraction rate.</div>
+        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;3) The eluent should be pre-warmed
+            to 65°C to increase the elution efficiency.</div>
+        <div class="article-content">3. Preparation Before Experiment:</div>
+        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1) Before using, ethyl alcohol
+            should be added to Buffer W2.</div>
+        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2) Make sure that the pipette tip
+            and microfuge tube are without nucleic acid contamination and no precipitations are in Buffer.</div>
+        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;3) Adjust the water bath to 65°C.
+        </div>
+        <div class="article-content">4. Protocol:</div>
+        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;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.</div>
+        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;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.</div>
+        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;3) Transfer the solution to EC and
+            centrifuge at 12,000 xg 22°C for 1 min and discard the collection tube.</div>
+        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;4) Add 700 μL Buffer W2 to EC and
+            centrifuge at 12000 xg 22°C for 1 min and discard the collection tube.</div>
+        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5) Repeat step 4.</div>
+        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;6) Transfer EC to the empty
+            collection tube and centrifuge at 12000 xg 22°C for 2 mins.</div>
+        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;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.</div>
+        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;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
+°C for 2 mins.</div>
+        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;9) Repeat step 8 for until all
+            solutions are eluted.</div>
+        <div class="article-title black-font" style="line-height: 32px;">Experiment 9: SDS-PAGE (Performed according to
+            Biochemistry Experiment and
+            Guidance of China Pharmaceutical University) </div>
+        <div class="article-content">1. Mix the following reagents: (TEMED is added last)</div>
+        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1) Monomer running gel</div>
+        <div class="img-wrap no-margin">
+            <img src="https://static.igem.org/mediawiki/2021/d/d7/T--Shanghai_Metro_Utd--experiments10.jpg" alt="">
+        </div>
+        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2) Monomer Stacking gel</div>
+        <div class="img-wrap no-margin">
+            <img src="https://static.igem.org/mediawiki/2021/4/45/T--Shanghai_Metro_Utd--experiments11.jpg" alt="">
+        </div>
+        <div class="article-content">2. Protocol:</div>
+        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;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.</div>
+        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;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.</div>
+        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;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.</div>
+        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;4) Pour the stacking gel above the
+            running gel.</div>
+        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;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.</div>
+        <div class="article-title black-font">Experiment 10: Protein Induction and Expression</div>
+        <div class="article-content">Protocol:</div>
+        <div class="article-content">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.</div>
+        <div class="article-content">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).</div>
+        <div class="article-content">3. Add IPTG to a concentration of 1 mmol/L.</div>
+        <div class="article-content">4. Induce expression at 37°C for 3 h.</div>
+        <div class="article-content">5. Centrifuged at 4°C at 4000 rpm for 10 min.</div>
+        <div class="article-content">6. Collect bacterial pellets.</div>
+        <div class="article-title black-font">Experiment 11: Preparation and dissolution of inclusion bodies</div>
+        <div class="article-content">Protocol:</div>
+        <div class="article-content">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.</div>
+        <div class="article-content">2. Vortex the bacterial suspension.</div>
+        <div class="article-content">3. Fissure the bacteria with a High Pressure Homogenizer.</div>
+        <div class="article-content">4. Centrifuge at 4℃ at 12000 rpm for 20 min and collect the precipitate.</div>
+        <div class="article-content">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.
+        </div>
+        <div class="article-content">6. Repeat step 5 and collect inclusion bodies. </div>
+        <div class="article-content">7. Conduct 15% SDS electrophoresis to analyze the bacterial liquid before and after
+            induction, inclusion bodies and supernatant after broken bacterial cells. </div>
+        <div class="article-content">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) </div>
+        <div class="article-content">9. Stir and dissolve in an anaerobic environment for 2 hours.</div>
+        <div class="article-content">10. Centrifuge at 4000r for 20 minutes to take the supernatant, which is the
+            denatured protein solution.</div>
+        <div class="article-title black-font">Experiment 12: Protein Refolding</div>
+        <div class="article-content">Protocol:</div>
+        <div class="article-content">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.</div>
+        <div class="article-content">2. After all the denaturation solution is added to the refolding buffer, continue
+            to stir the refolding buffer for about 1 h.</div>
+        <div class="article-content">3. Place the solution overnight at 4°C, and dilute to 2 L with ddH2O.</div>
+        <div class="article-title black-font">Experiment 13: Protein Purification</div>
+        <div class="article-content">Protocol:</div>
+        <div class="article-content">1. Pack SP Beads 6FF gel stored in 20% ethanol on the column (about 10 ml).</div>
+        <div class="article-content">2. Equilibrate 3 column volumes with buffer A (10 mM Tris-HCl, pH 8.0, 0.2 M NaCl).
+        </div>
+        <div class="article-content">3. Load the refolded protein solution filtered by a 0.22 μm filter membrane and
+            pass it through the column. </div>
+        <div class="article-content">4. Analyze the solution before and after passing the column by SDS-PAGE.</div>
+        <div class="article-content">5. Wash with buffer A for about one column volume and collect the washed solution.
+        </div>
+        <div class="article-content">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. </div>
+        <div class="article-content">7. Concentrate the eluted protein solution with an ultrafiltration tube and replace
+            it with ddH2O. </div>
+        <div class="article-content">8. Freeze the protein solution into a solid with liquid nitrogen.</div>
+        <div class="article-content">9. Lyophilized the protein solid with a freeze dryer to obtain a protein powder.
+        </div>
+        <div class="article-title black-font">Experiment 14: Protein Activity Verification I - RNA Extraction</div>
+        <div class="article-content">Protocol:</div>
+        <div class="article-content">1. Resuspend the HEK293-GFP cell pellet with 2 ml Trizol to a 15 ml centrifuge
+            tube.</div>
+        <div class="article-content">2. Leave it at room temperature for 5 min.</div>
+        <div class="article-content">3. Add 0.4 ml of chloroform and shake for 15 seconds.</div>
+        <div class="article-content">4. Leave it at room temperature for 5 min</div>
+        <div class="article-content">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.)
+        </div>
+        <div class="article-content">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.)</div>
+        <div class="article-content">7. Add an equal volume of pre-cooled isopropanol.</div>
+        <div class="article-content">8. Leave it at room temperature for 10 minutes to precipitate RNA.</div>
+        <div class="article-content">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.)</div>
+        <div class="article-content">10. Carefully remove the supernatant.</div>
+        <div class="article-content">11. Add 2 ml of 75% ethanol, blow with a gun several times.</div>
+        <div class="article-content">12. Centrifuge at 4°C at 7500 rpm for 5 min.</div>
+        <div class="article-content">13. Discard the supernatant and wait till the ethanol evaporates and dries.</div>
+        <div class="article-content">14. Add 40μl of enzyme-free water to resuspend all RNA, and measure the RNA
+            concentration and OD value with Nanodrop.</div>
+        <div class="article-content">15. Store the RNA at -80℃.</div>
+        <div class="article-title black-font">Experiment 15: Protein Activity Verification II - RNA Degradation</div>
+        <div class="article-content">Protocol:</div>
+        <div class="article-content">1. Replace the electrophoresis solution in the electrophoresis tank with a new one.
+        </div>
+        <div class="article-content">2. Prepare 3 reaction systems with PCR tubes.</div>
+        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1) 50 μl: 0.33 mmol/L Hepes, 0.33
+            mol/L NaCl, 883.82 ng/μl total RNA, 10 μmol/L rANG; </div>
+        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2) 50 μl: 0.33 mmol/L Hepes, 0.33
+            mol/L NaCl, 883.82 ng/μl total RNA, 1 μmol/L rANG; </div>
+        <div class="article-content">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;3) 50 μl: 0.33 mmol/L Hepes, 0.33
+            mol/L NaCl, 883.82 ng/μl total RNA, 0.2 mg/ml RNaseA.</div>
+        <div class="article-content">3. Metal bath at 37°C for 1 h.</div>
+        <div class="article-content">4. Take 50 μl and add 10 μl 6X gel loading to electrophoresis. (1% agarose gel
+            electrophoresis, 140 V, 40 min)</div>
+        <div class="article-title black-font">Experiment 16: Interaction study between heparin and Angiogenin</div>
+        <div class="article-content">Protocol:</div>
+        <div class="article-content">
+. Add 1mg/ml Heparan Sulfate to the transparent flat bottom 96-hole plate, cultivate for 2 hours. <br />
+. Wash the plate with PBS 3 times, 5 minutes each time. <br />
+. 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.<br />
+. Add anti-His (1:2000) coupled with HRP, react for 1 hour. Wash the plate with PBS 6 times.<br />
+. Add TMB, react for 30 minutes. Add 2 mol H2SO4. <br />
+. Use SpectraMax M5e to measure the absorption value.
+        </div>
+    </div>
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