The construction of CRISPReporter primarily involves common molecular experimental techniques, which are detailed on this page. Additionally, we have summarized the essential experimental protocols and PCR primer sequences used in our project, which can serve as a reference for others. If you have any inquiries or need further clarification, feel free to contact us at 2021302041074@whu.edu.cn.
Part 1: Fragments Obtaining
PCR
Taq PCR
Reaction system
- Total
- 2X Taq Master Mix
- Primer Forward
- Primer Reverse
- Template
- ddH2O
- 20(µl)
- 10
- 1
- 1
- 50ng
- Add up to 20µl
Program:
- 1
- 2
- 3
- 4
- 5
- 6
- Pre-Denaturation
- Denaturation
- Annealing
- Extension
- Jump to step 2, repeat for 30times
- Final Extension
- 95°C
- 95°C
- Tm-5°C
- 72°C
- Jump to step 2, repeat for 30times
- 72°C
- 30s (DNA) / 2min (Bacteria)
- 30s
- 30s
- 1min/Kb
- Jump to step 2, repeat for 30times
- 5min
HiFi PCR
Reaction system:
- Total
- HiFi PCR Enzyme
- 2 X Buffer
- dNTPs
- Primer Forward
- Primer Reverse
- Template
- ddH2O
- 50(µl)
- 1
- 25
- 1
- 2
- 2
- 100ng
- Add up to 50µl
Program:
- 1
- 2
- 3
- 4
- 5
- 6
- Pre-Denaturation
- Denaturation
- Annealing
- Extension
- Jump to step 2, repeat for 30times
- Final Extension
- 95°C
- 95°C
- Tm-5°C
- 72°C
- Jump to step 2, repeat for 30times
- 72°C
- 30s (DNA) / 2min (Bacteria)
- 30s
- 30s
- 1min/Kb
- Jump to step 2, repeat for 30times
- 5min
Touch Down PCR
Reaction system:
- Total
- 2X Taq Master Mix
- Primer Forward
- Primer Reverse
- Template
- ddH2O
- 20(µl)
- 10
- 1
- 1
- 50ng
- Add up to 20µl
Program:
- 1
- 2
- 3
- 4
- 5
- 6
- 7
- 8
- 9
- 10
- Pre-Denaturation
- Denaturation
- Annealing
- Extension
- Jump to step 2, repeat for 10 times, touch down 0.7°C per cycle
- Denaturation
- Annealing
- Extension
- Jump to step 6, repeat for 22 times
- Final Extension
- 94°C
- 94°C
- Tm°C
- 72°C
- Jump to step 2, repeat for 10 times, touch down 0.7°C per cycle
- 94°C
- Tm-7°C
- 72°C
- Jump to step 6, repeat for 22 times
- 72°C
- 30s (DNA) / 3min (Bacteria)
- 30s
- 30s
- 1min/Kb
- Jump to step 2, repeat for 10 times, touch down 0.7°C per cycle
- 30s
- 30s
- 1min/Kb
- Jump to step 6, repeat for 22 times
- 5min for fragments less than 10kb, 7min for fragments in 10-15kb
Error-Prone PCR
Reaction system:
- Total
- 2X Taq Master Mix
- Primer Forward
- Primer Reverse
- Template
- 50mM MnCl2
- ddH2O
- 50(µl)
- 25
- 2
- 2
- 50ng
- 1
- Add up to 50µl
Program:
- 1
- 2
- 3
- 4
- 5
- 6
- Pre-Denaturation
- Denaturation
- Annealing
- Extension
- Jump to step 2, repeat for 30times
- Final Extension
- 95°C
- 95°C
- Tm-3°C
- 72°C
- Jump to step 2, repeat for 30times
- 72°C
- 30s (DNA) / 2min (Bacteria)
- 30s
- 30s
- 1min/Kb
- Jump to step 2, repeat for 30times
- 5min
Annealing
Reaction system:
- Total
- Oligo I
- Oligo II
- 10X T4 Ligase buffer (NEB)
- T4 PNK (NEB)
- ddH2O
- 10 (µl)
- 100µM
- 100µM
- 1
- 1
- Add up to 10µl
Program:
- 37°C
- 95°C
- 4°C
- 30 min
- 5min and then ramp down to 25°C, at 5°C/min
- Hold until ready to proceed
Gel Extraction
- Cut out the target band minimizing extra gel.
- Put it in an Eppendorf tube (EP tube) and add 600μL QG.
- Incubate at 50°C until the gel is fully dissolved.
- Pour into the gel extraction column, spin through and discard the waste.
- Wash with 750μL PE buffer twice.
- Centrifuge at 13000rpm for 2min to dry.
- Elute with 50μl ddH2O.
Plasmid Preparation
- Pick a single colony, and cultivate in 5mL LB medium with antibiotic to saturation.
- Put the media into tubes repetitively.
- Centrifuge at 13000rpm for 1min.
- Discard the medium, and make the remaining pellet as dry as possible.
- Add 240μl SolutionBuffer I (mainly contains Glucose, NaCl) into the 1.5mL EP tube and resuspend the pellet.
- Add 240ul Buffer II (whose key component is NaOH) into the same EP tube.
- Mix it by gentle reversal 5 times until the solution gets clear.
- Add 350μl SolutionBuffer III (whose key component is acetate) into the same EP tube.
- Vortex the tube until the solution is uniform.
- Centrifuge at 12000rpm for 10min.
- Transfer the supernatant to a new mini-prep column.
- Centrifuge at 12000rpm for 1min and discard the flowthrough.
- Add 750μl SolutionBuffer PW (absolute alcohol containing) into the column.
- Centrifuge at 12000rpm for 1min and discard the flowthrough.
- Centrifuge again at 12000rpm for 2min to completely dry the column.
- Put the column into a new EP tube, and add 50μl ddH2O.
- Centrifuge at 12000rpm for 1min.
RNA Extraction
- Pick a single colony, and cultivate in 5mL LB medium with antibiotic to saturation.
- Put 1.5mL culture into a cooled 1.5mL EP tube.
- Centrifuge at 10000rpm for 3min.
- Discard the medium, add 200μl Bacteria RNA Plus Reagent at 95°C. Mix them together.
- Incubate at 95°C for 4 min.
- Add 1ml RNA isolator Total RNA Extraction Reagent, and place the mixture on ice for 5 min.
- Add 1/5 volume of pre-cooled chloroform to the above lysate. Vortex and shake for 15 sec, form an emulsion and let stand on ice for 5 min. Centrifuge at 4 °C at 12,000 rpm (13,800× g) for 15 min.
- Carefully remove the centrifuge tube. At this point, the solution is divided into three layers: the colorless upper layer, the white middle layer, and the red lower layer. Carefully pipette the upper aqueous phase into a new centrifuge tube.
- Add an equal volume of pre-cooled isopropanol and mix upside down. Let stand at -20°C for 10 min. Centrifuge at 4 °C at 12,000 rpm (13,800 × g) for 10 min. White deposits are usually visible.
- Carefully discard the supernatant and add 1 ml of pre-cooled RNase-free ddH2O prepared 75% ethanol. Wash the lid and wall well, flick the bottom of the tube, let the pellet suspension, and let stand at 4°C or ice for 3-5 min. Centrifuge at 4 °C at 12,000 rpm (13,800 × g) for 5 min. Discard the supernatant.
- Dry the precipitation in a clean environment at room temperature for 2-5 min.
- Add an appropriate amount of RNase-free ddH2O. Dissolve the precipitate, gently pipette until the precipitate is completely dissolved. After complete dissolution, take a small amount for testing, and store the rest at -80 °C.
Part 2: Subcloning
Restriction Cloning
One step cut digestion
Reaction system:
- Total
- Restriction Enzyme 1&2
- DNA
- 10X NEBuffer
- ddH2O
- 50 µl
- 1 µl each
- 1 µg
- 5 µl
- Add up to 50µl
Two steps cutdigestion
Reaction system 1:
- Total
- Restriction Enzyme 1
- DNA
- 10X NEBuffer
- ddH2O
- 50µl
- 1µl
- 1µg
- 5µl
- Add up to 50µl
Program 1:
- Incubation Time
- Incubation Temperature
- 15min
- 37°C
Reaction system 2:
- Total
- Restriction Enzyme 2
- Reaction system 1
- 10X NEBuffer
- ddH2O
- 150µl
- 3µl
- 50 µl
- 15µl
- 82 µl
Program 2:
- Incubation Time
- Incubation Temperature
- 15min
- 37°C
Ligation
Reaction system:
- 10X T4 DNA Ligase Buffer
- Vector DNA (4 kb)
- Insert DNA (1 kb)
- ddH2O
- T4 DNA Ligase
- 2 μl
- 50 ng (0.020 pmol)
- 37.5 ng (0.060 pmol)
- Add up to 20 μl
- 1 μl
Program: 25°C for 10min or 16°C overnight
Golden Gate Assembly
Reaction system:
- Vector
- Fragment
- T4 DNA ligase buffer
- T4 DNA ligase
- BsmB I / Bsa I
- H2O
- 1 μl
- 1 μl
- 1 μl
- 0.5 μl
- 0.5 μl
- 6 μl
Program
- 1
- 2
- 3
- 4
- 5
- 6
- 37°C
- 16°C
- Jump to Step1 and repeat for 30 times
- 50°C
- 80°C
- 10°C
- 3min
- 4min
- Jump to Step1 and repeat for 30 times
- 5min
- 15min
- forever
Gibson Assembly
Reaction system:
- Total
- 2× MultiF Seamless Assembly Mix
- Total DNA
- ddH2O
- 20µl
- 10µl
- 0.5 pmol (Vector:50ng)
- Add up to 20µl
Chemically Competent Cell Transformation
- Thaw chemically competent cells on ice.
- Add DNA (30ng), and pipette gently to mix.
- Let sit for 30 minutes on ice.
- Incubate cells for 45 seconds at 42℃.
- Chill on ice for 2 min.
- Add 1 ml LB at room temperature.
- Cultivate for 1 hour at 37℃, 150rpm.
- Centrifuge at 5000rpm for 2min, discard the supernatant to 100 μl and spread onto a plate with appropriate antibiotics.
- Grow overnight at proper temperature.
Part 3: Key Experiments
1.Induction of Editing on Genome
Plasmid pCas_Red_Δpoxb is transformed into the host by chemical transformation. The transformants carrying the editing plasmid are grown in 5 mL LB cultures with 50mg/L kanamycin at 30°C for 2 hours, and 2g/L arabinose is added to the culture. Then culture is incubated for approximately 24 hours at 30°C. At last, cells were diluted 100- fold and 20μL were plated on LB dishes containing 50mg/L kanamycin and 2g/L arabinose. Obtained colonies are identified by colony PCR with or without DNA sequencing.
2.Induction of Cascade Editing on Plasmids
Co-transform pCasop and relevant recording plasmid into Escherichia coli DH5[alpha] chemical component cells. Grow the transformants carrying the plasmids in 5 ml LB media with proper antibiotics at 30°C, 250 rpm for 12 hours. Then 100 µl bacteria liquid is transferred into 3 ml new LB media with proper antibiotics and cultures to OD 0.4-0.6. Then add 2g/L arabinose and cultivate for 17 hrs. Then add 3g/L IPTG and cultivate another 5hrs. After induction, dilute the cells to 100 folds and spread them on a plate with proper antibiotics.
3.Fluoroscopy
Place the dish in the gel image system, and turn on the UV lights. Observe the fluorescence from the window directly, or save the image for later measurement.
4.Reverse Transcription
Reaction system:
A. Prepare the first strand cDNA synthesis reaction solution
Prepare the following mixture in RNase-free centrifuge tubes:
- RNAse free ddH2O
- 5×HiScript 11 Select qRT SuperMix
- Random hexamers (50 ng/μl) or gene specific primers (2µM)
- Template RNA
- Add up to 20μl
- 4μl
- 1μl
- Total RNA:1pg-1vg
B. No RT Control reaction
No RT Control refers to a reverse transcription negative control reaction without reverse transcriptase to test RNA templates for genomic DNA residues. Prepare the following mixture in RNase-free centrifuge tubes:
- RNAse free ddH2O
- 5×Select No RT SuperMix
- Random hexamers (50 ng/μl) or gene specific primers (2µM)
- Template RNA
- Add up to 20μl
- 4μl
- 1μl
- Total RNA:1pg-1vg
Program:
- 50°C
- 85°C
- 15 min
- 5 sec
5.Key PCR primers
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