Team:Worldshaper-Shanghai/Protocols
Protocols
1. Get desired plasmid
1) Add 10 μL of TE into the dried plasmid, pipette up and down a few times, then wait 5 min until all the DNA dissolved.
2) Transform 3 μL of the plasmid into bacterial competent cells and plate them with corresponding antibiotic. Incubating in 37℃ overnight.
3) Choose a single colony and inoculate in LB broth with antibiotic. Grow cells at 37 ℃ for 16 h. separate the plasmid by mini-prep and preserve them in TE buffer.
2.Preparation for competent cell
(We use commercial E. coli DH5α competent cell in most condition)
1) Grow a single colony of E. coli in 2 mL LB overnight at 37 ℃ at 200 rpm
2) Inoculate 50 mL of LB liquid medium with 0.5mL of fresh bacterial overnight culture and grow cells at 37 ℃ for 2.5h. Cells should be harvested between an OD600 of 0.4 to 0.5.
3) Pour the bacteria solution into a 50 mL pre-cooled centrifuge tube and put the tube in an ice bath for 20 min. Then, use a desktop frozen centrifuge at a speed of 4000 rpm at 4 ℃ for 10 min. Discard the supernatant and invert the tube on a pre-ultraviolet irradiated and sterilize tissue in an ultra-clean table for 1 min.
4) Resuspend the cells in 30 mL of pre-cooled 0.1M CaCl2 by vortex oscillation in a 50 ml centrifuge tube, and put the cells on ice for 30 min. Centrifuged the cells at 4000 rpm at 4 ℃ for 10 min. Discard the supernatant and invert the tube on the sterilized tissue for 1 min.
5) Resuspend the cells in a final volume of 2 mL of cold 0.1M CaCl2 with 15% glycerol.
6) To freeze competent cells, aliquot in centrifuge tubes (100 μL/tube), and place tubes over dry ice until frozen. Store the cells at -80℃.
3. Bacterial Transformation
1) Take competent cells out of -80°C and thaw on ice (approximately 20-30 mins).
2) Mix 3 μL of DNA (usually 10 ng - 100 ng) into 100 μL of competent cells in a microcentrifuge tube. Gently mix by flicking the bottom of the tube with your finger a few times.
3) Incubate the competent cell/DNA mixture on ice for 20-30 mins.
4) Heat shock each transformation tube in a 42°C water bath for 90 secs.
5) Put the tubes back on the ice for 2 min.
6) Add 500 μL LB media (without antibiotic) to the bacteria and grow in 37°C shaking incubator for 1 h.
7) Plate 50 μL of the transformation onto a 10 cm LB agar plate containing the appropriate antibiotic.
8) Incubate plates at 37°C overnight.
4. Creating Bacterial Glycerol Stocks for Long-term Storage of Plasmids
1) Inoculating an overnight liquid culture of bacteria of interest.
2) Add 500 μL of the overnight culture to 500 μL of 50% glycerol in a 2 mL screw top tube or cryovial and gently mix.
3) Mark key information on the tube, including the name of each part, the carrier name, the strain name, operation date, name of personnel, etc.
4) Freeze the glycerol stock tube at -80°C. Subsequent freeze and thaw cycles reduce shelf life.
5. Plasmid Miniprep
1) Grow 2 mL overnight cultures from single colonies of bacteria containing the plasmid of interest.
2) Centrifuge in microfuge tube at 10,000 g for 30 sec.
3) Pour off the supernatant, being careful not to disturb the bacterial pellet.
4) Resuspend the pellet in 100 μL of cold Solution I. Vortex the solution for 2 mins or until all bacteria are fully resuspended.
5) Add 200 μL of Solution II and invert the tube carefully 5 times to mix the contents. The contents will become clear and thicker as the proteins and DNA are denatured.
6) Incubate solution on ice for 5 mins.
7) Add 150 μL of cold Solution III to each tube. Mix by inverting several times. A white precipitate will be formed which contains the bacterial proteins and genomic DNA.
8) Incubate the tube at room temperature for 5 mins.
9) Centrifuge the tube for 10 mins at 12,000 g.
10) Collect the supernatant into a new tube by pipetting. Add 700 μL of cold 100% ethanol to the solution to precipitate the plasmid DNA.
11) Centrifuge the tube for 10 mins at 12,000 g. Pour out the supernatant.
12) Wash the pellet with 70% ethanol.
13) Air dry the pellet for 10 mins.
14) Resuspend pellet with 20 μL of TE.
6. Polymerase Chain Reaction (PCR)
1) PCR component preparation
Thaw 10×Taq Buffer, dNTPs, template DNA, and primers at room temperature, and after that, place on ice. 10×Taq Buffer, dNTPs must be thoroughly mixed after thawing. Taq DNA Polymerase must place either on ice or in the refrigerator. When adding the Taq DNA Polymerase, it must be taken out from -20 degrees and put back immediately after adding.
2) Prepare PCR tubes and label them.
3) Preparation of PCR reaction. Add the following ingredients into the PCR reaction tube on ice:
Template DNA: 2 μL
10×Taq Buffer: 2 μL
dNTPs (2.5mM): 1.6 μL
Primer1 (10 μM): 0.4 μL
Primer2 (10 μM): 0.4 μL
Taq DNA Polymerase: 0.2 μL
ddH2O to a final volume 20 μL
4) Secure the tops to the PCR tubes, gently tap each tube to bring all the liquid to the bottom and centrifuge briefly.
5) PCR program
7. Agarose gel electrophoresis
1) Measure 1 g of agarose.
2) Mix agarose powder with 100 mL 1xTAE in a microwavable flask.
3) Microwave for 1-3 min until the agarose is completely dissolved.
4) Let agarose solution cool down to about 50°C, about 5 mins. And add 3 µl GelRed solution, and mix well by shaking.
5) Pour the agarose into a gel tray with the well comb in place. let sit at room temperature for 20-30 mins, until it has completely solidified.
6) place the agarose gel into the gel box. Fill gel box with 1xTAE until the gel is covered.
7) Add loading buffer to each of the DNA samples. Gently mix well with a pipette.
8) Carefully load a molecular weight ladder into the first lane of the gel. And carefully load test samples into the additional wells of the gel.
9) Run the gel at 100 V until the dye line is approximately 50-70% of the way down the gel. A typical run time is about 0.5-1 hours
10) Turn off power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.
11) Using the device that has UV light, visualize the DNA fragments. The fragments of DNA are usually referred to as ‘bands’ due to their appearance on the gel. Photographing the results.
8.Toehold switch Coloration (in vitro transcription/translation)
The protocol is based on S30 T7 High-Yield Protein Expression System from Promega
1) Remove the reagents from the freezer, and allow them to thaw on ice.
2) Set up the following reactions on ice in a DNase- and RNase-free PCR tube（20 μL reaction system):
3) Mix thoroughly by pipetting several times, then centrifuge in a microcentrifuge for 5 seconds to force the reaction mixture to the bottom of the tube.
4) Bring reaction to 37°C, and incubate the reactions for 3-4 h.
5) Check the mcherry signal with a white paper or with Biotek multiscan spectrum.
9. Reverse Transcription with TakaRa SYBR green RT reagent Kit
1) Remove the reagents from the freezer, and allow them to thaw on ice.
2) Set up the following reactions on ice in a DNase- and RNase-free PCR tube.
3) Secure the top to the PCR tube, gently tap each tube to bring all the liquid to the bottom and centrifuge briefly. And place reaction tubes in PCR machine.
4) Set program as follows: 37°C, 15min; 85°C, 5s(inactivate reverse transcriptase); 4°C, hold
5) Store the reverse-transcribed cDNA at -80°C for later use.
6) Semi-quantitative PCR: Take 1μL of cDNA as template and PCR amplify the target gene.
10. RNA enrichment from urine
1) Pipet 15 μL of Dynabeads MyOne Silane magnetic beads to the 5 mL sample, then add an equal volume of 100% ethanol to the RNA and Dynabeads mixture, and mix by inverting 5 times.
2) Incubate for 10 min at room temperature, invert 5 times every 2 minutes.
3) Apply a magnetic field to pull beads to the side of the tube. Carefully remove and discard the supernatant.
4) Re-suspend beads completely with 1mL of 70% ethanol. Then, transfer the magnetic beads and liquid to a 1.5ml microcentrifuge tube, and incubate for 5 minutes.
5) Apply a magnetic field to pull beads to the side of the tube. Carefully remove and discard the supernatant. Repeat wash a second time.
6) Air-dry the beads for 5 minutes at room temperature.
7) To elute, resuspend the beads completely in 15 μL of nuclease-free water and incubate for 5 min at room temperature. Apply the magnetic field to pull beads to the side of the tube and carefully collect supernatant without disturbing the beads into a clean tube. Then reserve it.
11. Standard curve
1) Preparation for Artificial Sample 1μg PCA3 mRNA( approximately 3.17 pmol) contains 1.77×10e12 molecules. Diluted to 1.5×10e6 copies / ml in 10 fold gradient with DEPC H2O. Then, dilute to the corresponding copy number (number of molecules) as needed.The number of copies were 0,50,100,200,300,400,500,800,1000,1500,2000 copies. 2 μ l PCA3 RNA with each different copy numbers above were added into 5ml synthetic urine and mixed as a sample.
2) Using magnetic beads to enrich RNA in synthetic urine and start reverse transcription.
3) The obtained cDNA was amplified by PCR.
4) Transcription and Translation:
Make up the 20 μl reaction system :
Toehold DNA(1μg)——————————————1μl
Standard PCR Product——————————2μl
S30 premix Plus———————————————8μl
S30 extract—————————————————7.2μl
DEPC H2O————————————————————1.8μl
Incubate at 37℃ for 4 hours. Take 10 μl reagent and dilute it to its one tenth; then add the solution to the 96 well plate . Then Use BioTek Multi-Mode Microplate Reader to examine the red fluorescence value.
12. ROC analysis
1) Adding 300 copies of PCA3 mRNA to 96 urine samples randomly
2) RNA enrichment, reverse transcription, PCR amplification and toehold reaction were performed in the same way as standard curve.
3) Use BioTek Multi-Mode Microplate Reader to examine the red fluorescence value.
2) Add 500 μL of the overnight culture to 500 μL of 50% glycerol in a 2 mL screw top tube or cryovial and gently mix.
3) Mark key information on the tube, including the name of each part, the carrier name, the strain name, operation date, name of personnel, etc.
4) Freeze the glycerol stock tube at -80°C. Subsequent freeze and thaw cycles reduce shelf life.
2) Centrifuge in microfuge tube at 10,000 g for 30 sec.
3) Pour off the supernatant, being careful not to disturb the bacterial pellet.
4) Resuspend the pellet in 100 μL of cold Solution I. Vortex the solution for 2 mins or until all bacteria are fully resuspended.
5) Add 200 μL of Solution II and invert the tube carefully 5 times to mix the contents. The contents will become clear and thicker as the proteins and DNA are denatured.
6) Incubate solution on ice for 5 mins.
7) Add 150 μL of cold Solution III to each tube. Mix by inverting several times. A white precipitate will be formed which contains the bacterial proteins and genomic DNA.
8) Incubate the tube at room temperature for 5 mins.
9) Centrifuge the tube for 10 mins at 12,000 g.
10) Collect the supernatant into a new tube by pipetting. Add 700 μL of cold 100% ethanol to the solution to precipitate the plasmid DNA.
11) Centrifuge the tube for 10 mins at 12,000 g. Pour out the supernatant.
12) Wash the pellet with 70% ethanol.
13) Air dry the pellet for 10 mins.
14) Resuspend pellet with 20 μL of TE.
Thaw 10×Taq Buffer, dNTPs, template DNA, and primers at room temperature, and after that, place on ice. 10×Taq Buffer, dNTPs must be thoroughly mixed after thawing. Taq DNA Polymerase must place either on ice or in the refrigerator. When adding the Taq DNA Polymerase, it must be taken out from -20 degrees and put back immediately after adding.
2) Prepare PCR tubes and label them.
3) Preparation of PCR reaction. Add the following ingredients into the PCR reaction tube on ice:
Template DNA: 2 μL
10×Taq Buffer: 2 μL
dNTPs (2.5mM): 1.6 μL
Primer1 (10 μM): 0.4 μL
Primer2 (10 μM): 0.4 μL
Taq DNA Polymerase: 0.2 μL
ddH2O to a final volume 20 μL
4) Secure the tops to the PCR tubes, gently tap each tube to bring all the liquid to the bottom and centrifuge briefly.
5) PCR program
7. Agarose gel electrophoresis
1) Measure 1 g of agarose.
2) Mix agarose powder with 100 mL 1xTAE in a microwavable flask.
3) Microwave for 1-3 min until the agarose is completely dissolved.
4) Let agarose solution cool down to about 50°C, about 5 mins. And add 3 µl GelRed solution, and mix well by shaking.
5) Pour the agarose into a gel tray with the well comb in place. let sit at room temperature for 20-30 mins, until it has completely solidified.
6) place the agarose gel into the gel box. Fill gel box with 1xTAE until the gel is covered.
7) Add loading buffer to each of the DNA samples. Gently mix well with a pipette.
8) Carefully load a molecular weight ladder into the first lane of the gel. And carefully load test samples into the additional wells of the gel.
9) Run the gel at 100 V until the dye line is approximately 50-70% of the way down the gel. A typical run time is about 0.5-1 hours
10) Turn off power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.
11) Using the device that has UV light, visualize the DNA fragments. The fragments of DNA are usually referred to as ‘bands’ due to their appearance on the gel. Photographing the results.
8.Toehold switch Coloration (in vitro transcription/translation)
The protocol is based on S30 T7 High-Yield Protein Expression System from Promega
1) Remove the reagents from the freezer, and allow them to thaw on ice.
2) Set up the following reactions on ice in a DNase- and RNase-free PCR tube（20 μL reaction system):
3) Mix thoroughly by pipetting several times, then centrifuge in a microcentrifuge for 5 seconds to force the reaction mixture to the bottom of the tube.
4) Bring reaction to 37°C, and incubate the reactions for 3-4 h.
5) Check the mcherry signal with a white paper or with Biotek multiscan spectrum.
9. Reverse Transcription with TakaRa SYBR green RT reagent Kit
1) Remove the reagents from the freezer, and allow them to thaw on ice.
2) Set up the following reactions on ice in a DNase- and RNase-free PCR tube.
3) Secure the top to the PCR tube, gently tap each tube to bring all the liquid to the bottom and centrifuge briefly. And place reaction tubes in PCR machine.
4) Set program as follows: 37°C, 15min; 85°C, 5s(inactivate reverse transcriptase); 4°C, hold
5) Store the reverse-transcribed cDNA at -80°C for later use.
6) Semi-quantitative PCR: Take 1μL of cDNA as template and PCR amplify the target gene.
10. RNA enrichment from urine
1) Pipet 15 μL of Dynabeads MyOne Silane magnetic beads to the 5 mL sample, then add an equal volume of 100% ethanol to the RNA and Dynabeads mixture, and mix by inverting 5 times.
2) Incubate for 10 min at room temperature, invert 5 times every 2 minutes.
3) Apply a magnetic field to pull beads to the side of the tube. Carefully remove and discard the supernatant.
4) Re-suspend beads completely with 1mL of 70% ethanol. Then, transfer the magnetic beads and liquid to a 1.5ml microcentrifuge tube, and incubate for 5 minutes.
5) Apply a magnetic field to pull beads to the side of the tube. Carefully remove and discard the supernatant. Repeat wash a second time.
6) Air-dry the beads for 5 minutes at room temperature.
7) To elute, resuspend the beads completely in 15 μL of nuclease-free water and incubate for 5 min at room temperature. Apply the magnetic field to pull beads to the side of the tube and carefully collect supernatant without disturbing the beads into a clean tube. Then reserve it.
11. Standard curve
1) Preparation for Artificial Sample 1μg PCA3 mRNA( approximately 3.17 pmol) contains 1.77×10e12 molecules. Diluted to 1.5×10e6 copies / ml in 10 fold gradient with DEPC H2O. Then, dilute to the corresponding copy number (number of molecules) as needed.The number of copies were 0,50,100,200,300,400,500,800,1000,1500,2000 copies. 2 μ l PCA3 RNA with each different copy numbers above were added into 5ml synthetic urine and mixed as a sample.
2) Using magnetic beads to enrich RNA in synthetic urine and start reverse transcription.
3) The obtained cDNA was amplified by PCR.
4) Transcription and Translation:
Make up the 20 μl reaction system :
Toehold DNA(1μg)——————————————1μl
Standard PCR Product——————————2μl
S30 premix Plus———————————————8μl
S30 extract—————————————————7.2μl
DEPC H2O————————————————————1.8μl
Incubate at 37℃ for 4 hours. Take 10 μl reagent and dilute it to its one tenth; then add the solution to the 96 well plate . Then Use BioTek Multi-Mode Microplate Reader to examine the red fluorescence value.
12. ROC analysis
1) Adding 300 copies of PCA3 mRNA to 96 urine samples randomly
2) RNA enrichment, reverse transcription, PCR amplification and toehold reaction were performed in the same way as standard curve.
3) Use BioTek Multi-Mode Microplate Reader to examine the red fluorescence value.
Worldshaper-Shanghai 2020
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