Team:Shanghai high school/Experiment
Experiments
Polymerase Chain Reaction
Aim: Amplify the target DNA fragment (12345) to a sufficient amount for experiment
Fragment Number:
N-lacZ
N-int
hERabr
C-int
C-lacZ
Preparation
We had divided the entire PCR amplification into four steps — we would first conduct the amplification of fragment 1+2 and fragment 3+4. After we successfully obtained fragment 1+2 and 3+4, we conducted the amplification of fragment 12+34. However, for fragment 1234+5, since both of the template fragments were too long, the PCR amplification was most likely to fail. Therefore, while conducting PCR amplification, we also developed an alternative plan — separately connect fragment 1234 and 5 to pET28a plasmid through homologous recombination
We prepared samples of PCR components for the amplification of 1+2 and 3+4, following the protocol listed below:
PCR Components Protocol
2x SF Buffer - 25 uL
dNTP (10 mM) - 1.2 uL
ddH2O - 21 uL
Phanta high-fidelity PCR enzyme - 1 uL
Template DNA (fragments) - 1 uL each
Primer PF - 0.3 uL
Primer PR - 0.3 uL
Conduct PCR amplification
We set up the temperature and cycles on the PCR amplifier following the protocol listed below
PCR Amplification Protocol
Step 1: 95℃ 3 min
Step 2: 95℃ 30 sec
Step 3: 54℃/56.5℃ 30 sec
Step 4: 72℃ 2.5 min
Step 5: Go to step 2 - 31 cycles
Step 6: 72℃ 5min
Step 7: 10℃ keep incubate
After the amplification was completed, we performed the electrophoresis to test if the length of result fragments reached our expectation.
Electrophoresis
Agarose Gel
For the performance electrophoresis, we prepared 130 ml of agarose mixture by adding 1.6 g of agarose powder into 130 ml of 1x TAE buffer in a conical flask and placed it into a microwave oven. After the mixture became homogeneous and transparent, we took the flask, evenly poured the solution into the casting tray, and inserted the comb, left it under room temperature until the solution was consolidated into a gel.
We then submerged the gel into running buffer in the electrophoresis chamber, added the nucleic acid dye and DNA loading buffer into each PCR tube and carefully transferred the marker and the dyed solution in each tube into its corresponding hole on the gel after centrifugation. After that, we put the cover back and turned on the power supply.
We cut off the power supply and collected the gel. Then we put it into the gel imaging system and documented the sizes of the bands.
In this case, the expected size of bands should be 981 bp (fragment 1+2) and 960 bp (fragment 3+4). Therefore, as it showed above, we could almost ensure that we had successfully obtained our target fragments by having both two bands about 1k in their size.
DNA Recovery
In order to progress our experiment, we had to recover the DNA fragments that were used in electrophoresis as template DNA to amplify the fragment (12+34).
Firstly, we carefully cut off the gel that contained our DNA fragments and collected them into a EP tube. Then we added buffer DE-A three times the value of the gel into the EP tubes and placed them into the heat machine until the solid gel was completely dissolved into liquid form. After that, we transferred the liquid into the adsorption column, which was later put into an EP tube, and added buffer DE-B half the value of buffer DE-A into the column. 12000r/min centrifugation was then processed and the effluent was removed. We then added 500 uL of buffer W1 and centrifuged it with 12000r/ min for one minute. After removing the effluent, we added 700 uL of buffer W2 into the column and centrifuged it for another minute. Penultimately, we removed the effluent and centrifuged it again for 2 minutes. And finally, after the ethanol absolute in buffer W2 has completely vaporized, we added 30 uL of Eluent, or distilled water, into the column and centrifuged it for 1 min with 12000r/min rotational rate. The solution left in the EP tube was our recovered DNA.
PCR Amplification
For the amplification of fragment 12+34 and later 1234+5, we repeated the steps above including preparation of PCR components, PCR conducting, electrophoresis, and DNA recovery. We successfully collected fragment 12345 at the end.
Homologous Recombination
After recovering the fragment 12345 from the gel, the team decided to use homologous recombination to connect our target DNA fragment to the empty plasmid pET28a. To ensure that we could successfully obtain the plasmid which carries our target DNA fragment, we design three possible combinations of homologous recombination:
Fragment 12345 recombines with plasmid pET28a
Fragment 1234 and fragment 5 recombine with plasmid pET28a at the same time
Fragment 1234 recombines with plasmid pET28a first
Incubation of E. coli
Aim: Transfer the product of homologous recombination into the competent cells DH5a. And as they reproduced, we could acquire a large amount of highly concentrated E. coli that carried our target plasmid for later experiments.
Preparation of Lysogeny Broth
Lysogeny Broth Protocol
Each Liter Consists of
Tryptone - 10 g
Yeast Extract - 5 g
Sodium Chloride - 10 g
Mineral Water - 960 g
Agar - 15 g (solid culture medium only)
Following the protocol, we prepared 900 ml of liquid culture medium in a large beaker and 300 ml of solid culture medium in another. We then put the beakers on a magnetic stirrer until the powder was completely dissolved in water. After that, we evenly distributed the liquid culture medium into three conical flasks and solid culture medium into one flask. Covered by a special sealing film, the flasks were sent to an autoclave for sterilization.
Plasmid Transformation
1. Unfreeze the E. coli competent cells on ice then add plasmid into the competent cells, keep the complex on ice for 30 min;
2. Heat shock the E. coli competent at 42oC water bath kettle for 1min;
3. Cool the E. coli competent immediately for 2min in ice;
4. Add no resistance LB liquid medium, keep E. coli competent regrowth in 37℃ shake cultivation for 45 min.
5.Centrifuge 12000rpm for 1 min, discard the liquid then spread the deposit on LB solid medium plate with antibiotics.
6.Put the plate in 37℃ constant temperature incubator for growth over night.
Petri Dishes Coating
Since the incubation of E. coli required sterile conditions, the process needed to be completed in a clean bench. Ozone ventilation and 75% ethanol sterilization of apparatus were also necessary before the experiment.
To prevent the E. coli that carried linear plasmid, or the target fragment that failed to combine with pET28a plasmid, from reproducing, we then added 0.1% of Kanamycin into the culture medium.
We evenly poured the culture medium into the petri dishes and waited until they had completely solidified. Then we removed 800ul supernatant from each EP tube, resuspended the strains at the bottom, and pipetted the remains on the culture medium. After that, we bent the pipette tips and used them to evenly coat the Petri dishes.
Finally, we put the cover back and reversely placed them into the incubator overnight under 37℃
Colony PCR
Aim: To test if we had successfully combined fragment 12345 and lacZ with plasmid pET28a through homologous recombination.
After the E.coli was incubated overnight, we select three Petri dishes that had the most E.coli colonies (the first one is painted with the E.coli contained our DNA fragment and connected with pET28a, the second one was with E.coli contained lacZ and pET28a, and the third one only had E.coli contained empty pET28a) and select several colonies in each dish. We then carefully transferred a small portion of each colony into PCR components mixture with pipette tips and centrifuged them with lower rotational rate.
Liquid Medium Culture
As we mentioned before, there were still remains of each colony that we selected to conduct PCR. We then used the pipette tips to transfer the remains from the Petri dishes to the tubes filled with liquid culture medium and added 0.1% of Kanamycin. An incubator shaker was used to incubate E. coli under 37℃ at 220rpm for 6 hours.
PCR
We drew 1 uL of fluid from each tube and transferred it into the PCR tube. The fluid was then diluted 100 times with double distilled water and mixed with PCR components according to the protocol.
After the same process of amplification, we have obtained the PCR products and then conducted electrophoresis.
Plasmid Extraction
Aim: 1. To transfer the plasmid into competent cell BL21 for later induction of protein expression.
2. Test the DNA sequence in order to eliminate the possibility of false positive.
To conduct the plasmid extraction, we had respectively prepared six CP3 columns for our target plasmid (fragment 12345 combined with pET28a), lacZ plasmid, and pET28a plasmid. They were then placed into six different recovery tubes and added with 500 uL of buffer BL. After 1 min of 12000 rpm centrifugation, we removed the effluent and placed the columns back in tubes.
We then pipetted 4ml E. coli fluid from each culture medium into a new EP tube (6 in total) and centrifuged them at 12000rpm for 1min. Subsequently, the supernatant was removed and we added 250 uL buffer P1, which contained Rnase A that would degrade the RNA remains. We resuspended the precipitate to break the lumps and ensure the bacteria could be sufficiently soaked in the solution. After that, we added 250 uL of buffer P2 into each EP tube and gently flipped it 6-8 times for NaOH to completely lyse the membrane. We then added 350 uL of buffer P3, gently flipped them for 6-8 times and centrifuged for 10 min after the precipitate emerged in the solution. This time supernatant was transferred into six CP3 tubes respectively and centrifuged to collect the precipitate. And to further wash off the impurities, we added 600 uL washing buffer PW, which contained absolute ethanol, and centrifuged them for 1 min. After removing the effluent, we centrifuged them for 2 min and waited until the ethanol absolute was completely vaporized. We eventually added 80 uL of Eluent into CP3 tubes, centrifuged them after placing under room temperature for 2 min, and the plasmid extract was eventually washed off into recovery tubes.
Electrophoresis
To ensure that we have successfully extracted the plasmid from DH5a E. coli, electrophoresis was performed to test if the plasmid was at expected length.
DNA Sequencing
We had sent a portion of our plasmid extract to DNA Sequencing Test in order to eliminate the false positives.
Since the heterozygous peaks are most likely to appear in the first 30 bp, instead of starting sequencing from fragment one, we advanced the start to T7 promoter on pET28a plasmid.
BL21 E. coli Incubation
We transferred EP tubes into the clean bench as the E. coli fluid became turbid. After sufficient sterilization of the clean bench and apparatus with ozone gas and ultraviolet light, E. coli fluid was respectively coated on the Petri dishes. And we then placed the Petri dishes reversely in the incubator overnight.
The second day we selected the best-incubated colonies and transferred them into the tubes which contained liquid culture medium with 0.1% Kanamycin. Then tubes were placed into the incubator shaker at 37℃ and 210 rpm for 6 hours.
IPTG Induction and Protein Purification
Only for the E. coli that carried our target fragment or lacZ
The incubated E. coli fluid was then added into conical flasks filled with culture medium and a small portion was collected as a control for protein electrophoresis. We added 0.1% of Kanamycin and into the flasks and kept them in the shaker for another six hours.
Preparation
In the process of protein purification, we had prepared three buffers - lysis buffer, binding buffer, and washing buffer - that consisted of the same components but were used differently in procedure.
Buffers Protocol
Each 500 ml consists of
Tris HCl (pH 8.0) - 25 ml
NaCl - 50 ml
Glycerol - 25 ml
DTT - 0.5 ml
ddH2O - 400 ml
Protein Purification
The induced E. coli fluid in each flask was evenly distributed into 12 centrifuge tubes (24 tubes in total) and centrifuged for 10 min at 6500 rpm. (Note: we also collected 1 ml of the fluid from each conical flask as a sample.)
The tubes were then placed on ice. For every 12 tubes with the E. coli that carried the same plasmid, we removed supernatant and added 15 ml of lysis buffer into the first tube. (Note: In this step, we collected 1 ml of the solution as a sample) After resuspending the precipitate, we pipetted the solution into the second tube and resuspended the precipitate again. Repeating this process, we finally collected everything into one tube and 65 uL of PMSF was then added to prevent the protein from being hydrolyzed by protease. Subsequently, we placed the tube into the ultrasonic cell disruptor and set the power to 30%. The working time of cell disruption was 10 min, then we distributed the product into several 1.5 ml EP tubes and centrifuged them at 4C and 13000 rpm for 50 min.
Nickel Column Affinity Chromatography
Aim: the nickel column could bind with the anti-6*His tag monoclonal antibody that was carried by our target protein. Therefore, the protein would be separated from the impurities and then washed off by imidazole, which had better affinity to nickel.
After centrifugation, we collected the supernatant in EP tubes (Note: we kept 1 ml of supernatant from each group as a sample) and transferred it into the nickel column after adding the binding buffer. The nickel column was fixed on a centrifuge tube so the flew through could be collected and used to repeat the same process for 3 times. (Note: 1 ml of flew through solution was collected as a sample) At this point, our target protein had already been blinded with the nickel column. And then we added a decent amount of washing buffers with 20 mmol of imidazole to ensure that the impure protein was completely washed off.(Note: 1 ml of flew through was collected as a sample) Finally, after removing the flew through solution from the centrifuge tube, we added the washing buffer with 200 mmol of imidazole into the column, and the target protein was collected into the tube.
SDS-PAGE Gel Electrophoresis.
Aim: To identify our target protein.
Preparation
Separating Gel Protocol
Buffer - 5 ml
Gel solution - 5 ml
Coagulant - 100 ml
Stacking Gel Protocol
Buffer - 1.5 ml
Gel solution - 1.5 ml
Coagulant - 30 ml
Following the protocol, we successfully prepared the liquid form of separating gel. We then carefully poured the gel into the casting tray until it reached the bottom of the comb. Double distilled water was added to even out the surface and the gel was placed into the thermotank for 30min at 37℃ After that, we poured out the double distilled water and added stacking gel solution into the casting tray. We carefully inserted the comb and placed the gel into the thermotank under 37℃ until it was solidified.
Electrophoresis
For the samples that we collected throughout the entire process, we transferred them into different EP tubes and placed them into the metal bath under 100C for 5 min to lyse the membrane and collect the protein.
After the stacking gel was solidified, we slowly removed the comb and placed the gel into the electrophoresis chamber and poured it in the running buffer. Subsequently, we added the SDS-PAGE loading buffer into each tube and then carefully transferred the protein in each EP tube into the holes on gel. After that, we connected the electrodes and then turned on the power supply.
At the same time, we also need to prepare the dye for later band analysis.
Following the protocol, we had made 300ml of Coomassie brilliant blue solution. Then we poured the solution into the electrophoresis chamber which carried our product and placed the chamber into the shaking incubator for 30 minutes (in order to dye the gel evenly). After that, we took out the chamber and placed it under room temperature for 10 minutes. The decoloring buffer was added to the gel, and then we waited another 20 minutes until the Coomassie blue was completely washed off. The blue bands remained on the gel were out results.
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