All of our PCR amplifications follow the instructions of PrimeSTAR® HS DNA Polymerase(Takara).
Reagent | Volume/Amount | Final Concentration |
---|---|---|
5 × PrimeSTAR Buffer(Mg2+ Plus) | 10 μL | 1x |
dNTP Mixture(2.5mM each) | 4 μL | 200 μM each |
Primer 1 | 10-15 pmol | 0.2-0.3 μM |
Primer 2 | 10-15 pmol | 0.2-0.3 μM |
Template | <200 ng | Plasmid DNA 10 pg-10 ng |
PrimeSTAR HS DNA Polymerase(2.5U/μl) | 0.5 μL | 1.25 U/50 μL |
ddH2O | Up to 50 μL | - |
Temperature | Time | ||
---|---|---|---|
Initial Denaturation | 98℃ | 1min | |
Denaturation | 98℃ | 10s | |
Annealing | 55℃ | 5s | x35 |
Extension | 72℃ | 1min/kb | |
Final Extension | 72℃ | 2min | |
Hold | 4℃ | ∞ |
Note: The process of adding reagents should be done on ice. Enzymes should be added last. The system should be vortexed to mix after configuration and centrifuged instantly before PCR amplification. Experimental conditions should be adjusted in detail according to the actual situation.
All of our PCR amplifications follow the instructions of PrimeSTAR® HS DNA Polymerase(Takara).
Reagent | Volume/Amount | Final Concentration |
---|---|---|
5 × PrimeSTAR Buffer(Mg2+ Plus) | 10 μL | 1x |
dNTP Mixture(2.5mM each) | 4 μL | 200 μM each |
Primer 1 | 10-15 pmol | 0.2-0.3 μM |
Primer 2 | 10-15 pmol | 0.2-0.3 μM |
Template | <200 ng | Plasmid DNA 10 pg-10 ng |
PrimeSTAR HS DNA Polymerase(2.5U/μl) | 0.5 μL | 1.25 U/50 μL |
ddH2O | Up to 50 μL | - |
Temperature | Time | ||
---|---|---|---|
Initial Denaturation | 98℃ | 1min | |
Denaturation | 98℃ | 10s | |
Annealing | 55℃ | 5s | x35 |
Extension | 72℃ | 1min/kb | |
Final Extension | 72℃ | 2min | |
Hold | 4℃ | ∞ |
Note: The process of adding reagents should be done on ice. Enzymes should be added last. The system should be vortexed to mixed after configuration and centrifuged instantly before PCR amplification. A small portion of a single colony is gently picked with the tip of the gun, and then the colony-soaked tip is placed into the reaction solution and stirred a bit to allow the colony to enter the PCR system. DNA template is from the colony in this reaction. Experimental conditions should be adjusted in detail according to the actual situation.
All of our assembly solutions use the Golden Gate assembly solution provided in SEVA 3.1, enabling interoperability of DNA assembly among the SEVA, BioBricks, and Type IIS restriction enzyme standards.
Reagent | Volume |
---|---|
10x Buffer for T4 DNA ligase with 10mM ATP (NEB) | 18 μL |
BSA (20mg/ml) | 1 μL |
DpnI (NEB) | 1 μL |
T4 DNA ligase (NEB) | 10 μL |
BsaI HFv2 (NEB) | 12 μL |
Total | 42 μL |
Reagent | Volume | Final Concentration |
---|---|---|
Golden Gate Master Mix | 2 μL | - |
DNA for assembly | 6 μL | The concentration of all DNA sequences to be assembled in the final system should be 1 nM |
Temperature | Time | ||
---|---|---|---|
Initial Denaturation | 37℃ | 20min | |
Ligase | 16℃ | 4min | x30 |
Digest | 37℃ | 20min | x30 |
50℃ | 10min | ||
Inactivation | 80℃ | 10min | |
Hold | 4℃ | ∞ |
Note: The process of adding reagents should be done on ice. Enzymes should be added last. The system should be vortexed to mixed after configuration and centrifuged instantly before PCR amplification.
The in vitro transcription protocol follows the instructions for the T7 High Yield RNA Synthesis Kit (Yeasen).
Reagent | Volume/Amount | Final Concentration |
---|---|---|
CTP (100mM each) | 2 μL | 10 mM |
GTP (100mM each) | 2 μL | 10 mM |
ATP (100mM each) | 2 μL | 10 mM |
UTP (100mM each) | 2 μL | 10 mM |
10x Transcription Buffer | 2 μL | 1x |
Template DNA | 1 μg | - |
T7 RNA Polymerase Mix | 2 μL | - |
RNase-free H2O | Up to 20 μL | - |
Mix the above reaction solution, briefly centrifuge to the bottom of the tube, and incubate at 37℃ for 4~8 hours.
Gel electrophoresis is used to test whether PCR is successful or not.
Reagent | Volume/Amount | Final Concentration |
---|---|---|
Agarose | 2 g | 1% |
1x TAE Buffer | 200 mL | 1 |
Gel volume can be increased or decreased according to actual needs. More buffer can be added when configuring the gel solution to offset heat evaporation losses.
We use the NorthernMax™ Kit (Thermo Fisher) to do this experiment.
Reagent | Volume/Amount | Final Concentration |
---|---|---|
Agarose | 1 g | 1% |
1x MOPS Buffer | 82 mL | |
Formaldehyde | 18 mL |
Gel volume can be increased or decreased according to actual needs. More buffer can be added when configuring the gel solution to offset heat evaporation losses.
We follow the instructions for the TIANprep Rapid Mini Plasmid Kit (TIANGEN).
Experimental conditions should be adjusted in detail according to the actual situation. The amount of plasmid extracted is related to the bacterial culture concentration, plasmid copy number, and other factors. The adsorption and elution time can be extended appropriately to improve the extraction efficiency.
We follow the instructions for the EndoFree Maxi Plasmid Kit (TIANGEN).
Experimental conditions should be adjusted in detail according to the actual situation. The amount of plasmid extracted is related to the bacterial culture concentration, plasmid copy number, and other factors. If the extracted plasmid is a low-copy plasmid or a large plasmid larger than 10 kb, the amount of bacteriophage used should be increased, and the amount of P1, P2, and P4 should be increased proportionally; the elution buffer is recommended to be preheated in a 65-70°C water bath. The adsorption and elution time can be extended appropriately to improve the extraction efficiency.
We follow the instructions for the TIANgel Midi Purification Kit (TIANGEN).
All centrifugation steps were performed at room temperature using a bench-top centrifuge. Experimental conditions should be adjusted in detail according to the actual situation. If the gel weighs 0.1g, its volume can be considered as 100 μl, so 100 μl of PN should be added.
We follow the instructions for the RNAclean Kit (Tiangen).
Add ethanol to buffer RW in advance. Add β-mercaptoethanol to RK to a final concentration of 1% before first use. Experimental conditions should be adjusted in detail according to the actual situation.
We follow the instructions for the His-tag Protein Purification Kit, His-Tag Protein Purification Kit with IDA-Ni Magnetic Agarose Beads, and SUMO Protease (Beyotime).
Reagent | Volume/Amount |
---|---|
10X Reaction Buffer –/+ Salt | 20 μL |
SUMO-tag Protein (20μg) | Y μL |
SUMO Protease (10U/μl) | 1μL |
H2O | Up to 200 μl |
Enzymatic digestion at 25℃ for 1.5h
Experimental conditions should be adjusted in detail according to the actual situation.
Experimental conditions should be adjusted in detail according to the actual situation.
Reagent | Volume/Amount |
---|---|
RO H2O | 4 ml |
30% Acrylamide mixture | 3.1 ml |
1.5M Tris-HCl | 2.5 ml |
10% SDS | 0.1 ml |
10% Ammonium persulfate solution | 0.1 ml |
TEMED | 0.004 ml |
Total | 10 ml |
Reagent | Volume/Amount |
---|---|
RO H2O | 1.4 ml |
30% Acrylamide mixture | 0.33 ml |
1.0M Tris-HCl | 0.25 ml |
10% SDS | 0.02 ml |
10% Ammonium persulfate solution | 0.02 ml |
TEMED | 0.002 ml |
Total | 2 ml |
Experimental conditions should be adjusted in detail according to the actual situation.
Reagent | Volume/Amount |
---|---|
RO H2O | 4 ml |
30% Acrylamide mixture | 3.1 ml |
1.5M Tris-HCl | 2.5 ml |
10% SDS | 0.1 ml |
10% Ammonium persulfate solution | 0.1 ml |
TEMED | 0.004 ml |
Total | 10 ml |
Reagent | Volume/Amount |
---|---|
RO H2O | 1.4 ml |
30% Acrylamide mixture | 0.33 ml |
1.0M Tris-HCl | 0.25 ml |
10% SDS | 0.02 ml |
10% Ammonium persulfate solution | 0.02 ml |
TEMED | 0.002 ml |
Total | 2 ml |
Reagent | Volume/Amount |
---|---|
Non-fat milk | 2.5 g |
TBST | 50 ml |
Experimental conditions should be adjusted in detail according to the actual situation. Secondary antibody we used is HRP. Sponge and filter paper need to be soaked for 30min in advance.
All of our PCR amplifications follow the instructions of Trans1-T1 Phage Resistant Chemically Competent Cell (Transgen).
Reagent | Volume/Amount |
---|---|
Trans1-T1 Phage Resistant Chemically Competent Cell | 40 μL |
Plasmids to be transformed | X ng |
The process of adding reagents should be done on ice. Experimental conditions should be adjusted in detail according to the actual situation.
Restriction enzyme digestion is used to verify the success of PCR amplification.
Reagent | |
---|---|
Cut Buffer | Follow the instructions for the enzyme |
Enzyme | |
DNA | |
ddH2O |
The process of adding reagents should be done on ice. Enzymes should be added last. Experimental conditions should be adjusted in detail according to the actual situation.
Reagent | Volume/Amount |
---|---|
Yeast Extract | 5g |
Tryptone | 10g |
NaCl | 10g |
NaOH | Until pH=7.0 |
H2O | Up to 1 L |
Autoclave for 20 minutes after mixing.
Reagent | Volume/Amount |
---|---|
Yeast Extract | 5g |
Tryptone | 10g |
NaCl | 10g |
Agar powder | 1.5g |
NaOH | Until pH=7.0 |
H2O | Up to 1 L |
Autoclave for 20 minutes after mixing. Add antibiotics when the temperature drops to 55°C.
Reagent | Volume/Amount | |
---|---|---|
Yeast Extract | 23.6 g | Solution 1 (with 900ml H2O) |
Tryptone | 11.8 g | |
Glycerol (80%) | 4 ml | |
K2HPO4 | 9.4 g | Solution 2 (with 100ml H2O) |
KH2PO4 | 2.29 g | |
Total | 1 L |
After preparing solutions 1 and 2, autoclave them. When solution 1 is cooled to below 60°C, pour in solution 2. Add antibiotics to the medium after cooling.
Reagent | Volume/Amount | |
---|---|---|
Yeast Extract | 5g | |
Tryptone | 20 g | SOB |
NaCl | 0.5 g | |
KCl(250mmol/L) | 10 mL | |
MgCl2(2mol/L) | 5 mL | |
Glucose(1mol/L) | 20 mL | |
Total | 1 L |
SOB was prepared first. Autoclaved and cooled it to below 60°C. Then add glucose (decontaminated) to it.
50× TAE solution was diluted to obtain 1× TAE solution for gel electrophoresis.
Reagent | Volume/Amount |
---|---|
Tris (hydroxymethyl)aminomethane(Tris) | 242 g |
Na2EDTA•2H2O | 37.2 g |
Glacial acetic acid | 57.1 ml |
Reverse osmosis H2O | Up to 1L |
Mix the above reagents.
Reagent | Volume/Amount |
---|---|
OPti-Medium | 950 μl |
RNA iMax | 5 μl |
Mimic-21-5p | 5 μl |
*Wait 5min after mixing OPti-Medium and RNA iMAX. Then add mimic-21-5p to it and wait for 20min.
Reagent | Volume/Amount |
---|---|
Cells | 8*10^5 |
DMEM(- -) | 100 μl |
Add 100μl of solution 1 to solution 2.
Note: Experimental conditions should be adjusted in detail according to the actual situation. Mimic-21-5p is consist of 1OD powder dissolved with 125 μl DEPC H2O.
We performed an invasion assay to verify that miR-21-5p has a role in promoting breast cancer cell invasion.
Note: Experimental conditions should be adjusted in detail according to the actual situation.
We performed this experiment to verify that miR-21-5p promotes the proliferation of breast cancer cells.
Note: Experimental conditions should be adjusted in detail according to the actual situation.
To determine the best crRNA that we designed, we used four crRNA sequences designed by us for FAM-BHQ RNA probe cleavage, so that we can test the collateral cleavage activity of different crRNAs stimulated by the guidance of a single-stranded RNA like miRNAs.
Reagent | NC | CR - 1 | CR - 2 | CR - 3 | CR - fl |
---|---|---|---|---|---|
ddH2O | 12 μL | 10 μL | 10 μL | 10 μL | 10 μL |
Reagent | Quantity |
---|---|
miRNA-21-5p (1μM) | 2 μL |
Fluorescence Reporter (1μM) | 4 μL |
ddH2O | 14 μL |
Formulate Reaction System 1 and Reaction System 2 on ice, centrifuge and mix, blow to homogeneity, then sample into a ninety-six well plate and analyze in an immunoenzymology analyzer.
We established different concentration gradients (miRNA concentrations of 0nM, 25nM, 50nM, 100nM) to test the optimal miRNA concentration for activating the CRISPR-Cas system.
Reagent | NC | CR - 1 | CR - 2 | CR - 3 | CR - fl |
---|---|---|---|---|---|
ddH2O | 12 μL | 10 μL | 10 μL | 10 μL | 10 μL |
10 × Cas13a Reaction Buffer | 4 μL | 4 μL | 4 μL | 4 μL | 4 μL |
RNase inhibitor (40U) | 2 μL | 2 μL | 2 μL | 2 μL | 2 μL |
crRNA-21-5p (0.4μM) | 0 μL | 2 μL crRNA-21-5p-1 | 2 μL crRNA-21-5p-2 | 2 μL crRNA-21-5p-3 | 2 μL crRNA-21-5p-fl |
LwaCas13a protein (2μM) | 2 μL | 2 μL | 2 μL | 2 μL | 2 μL |
Total | 20 μL | 20 μL | 20 μL | 20 μL | 20 μL |
Reagent | NC | miR(10nM) | miR(25nM) | miR(50nM) | miR(100nM) | miR(200nM) |
---|---|---|---|---|---|---|
ddH2O | 16 μL | 12 μL | 6 μL | 14 μL | 12 μL | 8 μL |
miRNA-21-5p | 0 μL | 4 μL * 0.1 μM | 10 μL * 0.1 μM | 2 μL * 1 μM | 4 μL * 1 μM | 8 μL * 1 μM |
Fluorescence Reporter (1μM) | 4 μL | 4 μL | 4 μL | 4 μL | 4 μL | 4 μL |
Total | 20 μL | 20 μL | 20 μL | 20 μL | 20 μL | 20 μL |
Formulate Reaction System 1 and Reaction System 2 on ice, centrifuge and mix, blow to homogeneity, then sample into a ninety-six well plate and analyze in an immunoenzymology analyzer.
We tested the changes in fluorescence intensity induced by different concentration gradients of Reporter RNA (0nM, 100nM, 125nM, 250nM, 500nM) over time.
Reagent | Quantity |
---|---|
ddH2O | 10 μL |
10 × Cas13a Reaction Buffer | 4 μL |
RNase inhibitor (40U) | 2 μL |
crRNA-21-5p (0.4μM) | 2 μL |
LwaCas13a protein (2μM) | 2 μL |
Total | 20 μL |
Reagent | NC | Reporter (100nM) | Reporter (125nM) | Reporter (167nM) | Reporter (250nM) | Reporter (500nM) |
---|---|---|---|---|---|---|
ddH2O | 18 μL | 14 μL | 13 μL | 14 μL | 16 μL | 16 μL |
miRNA-21-5p | 2 μL | 2 μL | 2 μL | 2 μL | 2 μL | 2 μL |
Fluorescence Reporter (1μM) | 0 μL | 4 μL * 1 μM | 5 μL * 1 μM | 4 μL * 5/3 μM | 2 μL * 5 μM | 2 μL * 10 μM |
Total | 20 μL | 20 μL | 20 μL | 20 μL | 20 μL | 20 μL |
Formulate Reaction System 1 and Reaction System 2 on ice, centrifuge and mix, blow to homogeneity, then sample into a ninety-six well plate and analyze in an immunoenzymology analyzer.
We established different concentration gradients (miRNA concentrations of 0nM, 5nM, 25nM, 50nM, 100nM) to demonstrate the feasibility of the test strip and the discrimination of the test strip between high and low concentrations of miRNA.
Reagent | Volume |
---|---|
ddH2O | 5 μL |
10 × Cas13a Reaction Buffer | 2 μL |
RNase inhibitor (40U) | 1 μL |
crRNA-21-5p (0.4μM) | 1 μL |
LwaCas13a protein (2μM) | 1 μL |
Total | 10 μL |
Reagent | NC | miR(5nM) | miR(25nM) | miR(50nM) | miR(100nM) |
---|---|---|---|---|---|
10 × Cas13a Reaction Buffer | 2 μL | 2 μL | 2 μL | 2 μL | 2 μL |
RNase inhibitor (40U) | 1 μL | 1 μL | 1 μL | 1 μL | 1 μL |
miRNA-21-5p (1μM) | 0 μL | 0.1 μL | 0.5 μL | 1 μL | 2 μL |
Reporter (2μM) | 2.4 μL | 2.4 μL | 2.4 μL | 2.4 μL | 2.4 μL |
ddH2O | Up to 10 μL | Up to 10 μL | Up to 10 μL | Up to 10 μL | Up to 10 μL |
After mixing the working solution and the sample solution, the reaction solution was obtained, and the reaction droplets were added to the sample pad of the test strip. The result was recorded in 3 minutes.
Primer Name | Sequence |
---|---|
Primer Loop R | AGGTCTCTCCCTTCGTTTTTGGGGTAGTCTAAATCTATAGTGAGTCGTATTACTCTAGAAGCGGCCGCGAATTC |
Primer Loop F | AGGTCTCAGAAGATCCTTTGATCTTTTCTACGGGGTCTG |
Primer Spacer R | AGGTCTCACTTCTTGAGATCCTTTTTTTCTGCGCGTAAT |
Primer Spacer F(full length) | AGGTCTCAAGGGGACTAAAACTCAACATCAGTCTGATAAGCTACAGAGAAGAGCTACTAGTAGCGGCCGCTGCAG |
Primer Spacer F(1) | AGGTCTCAAGGGGACTAAAACCAACATCAGTCTGATAAGCTCAGAGAAGAGCTACTAGTAGCGGCCGCTGCAG |
Primer Spacer F(2) | AGGTCTCAAGGGGACTAAAACAACATCAGTCTGATAAGCTACAGAGAAGAGCTACTAGTAGCGGCCGCTGCAG |
Primer Spacer F(3) | AGGTCTCAAGGGGACTAAAACTCAACATCAGTCTGATAAGCCAGAGAAGAGCTACTAGTAGCGGCCGCTGCAG |
Primer Backbone R | AGGTCTCACTCTAGAAGCGGCCGCGAATTC |
Primer Backbone F | AGGTCTCATACTAGTAGCGGCCGCTGCAG |
Primer LacI R | AGGTCTCAAGTAACAGTCATAAGTGCGGC |
Primer LacI F | AGGTCTCACACCGATGGGGAAGATCG |
Primer Cas R | AGGTCTCAGGTGATGTCGGCGATATAGG |
Primer Cas F | AGGTCTCAAGAGCAAAAAACCCCTCAAGAC |
Primer VR | ATTACCGCCTTTGAGTGAGC |
Primer VF2 | TGCCACCTGACGTCTAAGAA |
Primer CHECK R | CACGAGAAGTACAAGATCCGCGAGTAC |
Primer CHECK F | AGTACTCGCGGATCTTGTACTTCTCGTG |