1. PCR
Prepare:
l Materials
|
Reagent |
Volume/mass |
|
Template plasmid |
4μL |
|
PrimeSTAR Max Premix |
100μL |
|
Primer-F |
8μL |
|
Primer-R |
8μL |
|
ddH2O |
To 200μL |
*PrimeSTAR Max Premix includes DNA polymerase, dNTP and buffer.
|
Apparatus |
Amount |
|
PCR thermal cycler |
1 |
|
Pipette |
1 |
|
Microcentrifuge tube |
3 |
l Procedure
1. Configurate the PCR system. Add s into tube
2. Divide 200 μL system to 4*50 μL system
3. Put 4 tubes into PCR thermal cycler.
4.Set PCR program 95℃ 5 min；95℃ 30 s，55℃ 30 s，72℃ 30，Cycles 72℃ 10 min 4℃∞。
Agarose Gel Electrophoresis
l Materials
|
Reagents |
Volume/mass |
|
Agarose |
1g |
|
TAE Buffer |
100mL |
|
Ts-GelRed |
10μL (10000X) |
|
DNA Loading Buffer |
10X in template |
|
Marker |
5μL |
|
Template |
-- |
|
Apparatus |
Amount |
|
Conical flask |
1 |
|
Pipette |
1 |
|
Weighing paper |
1 piece |
|
Electronic balance |
1 |
|
Microwave oven |
1 |
|
Mold for creating gel |
1 |
|
Electrophoresis chamber |
1 |
|
UV transilluminator |
1 |
|
Scalpel |
1 |
l Procedure
Making gel
1. Use a balance and a digital scale to get the exact amount of reagent that we need.
2. Add all the reagents into a conical flask
3. Place the conical flask into microwave oven until the reagents in the conical flask turn clear.
4. Placed the beaker under water and slowly shook it so that the every part of the mixture can cooled down evenly.
5. Add 10μL nuclei acid dye by using a pipette( be careful as it is poisonous)
6. Put the gel into the mold and insert a comb into the gel. Wait until the gel is solidified.
Electrophoresis
1. Take out the solidified gel and place it in the electrophoresis chamber.
2. Add the loading buffer to the sample.
3. Add 5μL marker and samples in the hole of the gel.
4. Start the electrophoresis and wait for about 30 minutes.
5. After the agarose gel electrophoresis done, using UV light to detect the DNA ladder and cut down the gel with DNA,
6. Use Gel doc to take the photo of DNA stripe
2. Recycle the gel
l Goal: purify the DNA sequence that we needed for latter experiment.
l Material
Reagent
agarose gel
Binding buffer
deionized water: 20µl
wash solution:500µl
b. Apparatus
pipette
pipette tips
metal bath instrument
l Procedures
1.Add agarose gel and binding buffer into one microcentrifuge tube.
2. Place the tube in water bath at 50°C for 10 minutes.
3. Transfer the solution into the spin column.
4. Centrifuge the column at 12000rpm for 1 minute.
5. Add 700μL wash buffer into the spin column and centrifuge.
6. Repeat steps 5 for 2 times.
7. Centrifuge the tube at 12000rpm for 2 minutes
8. Put the spin column into a new microcentrifuge tube.
9. Add 20μL water to the column membrane and centrifuge
3. In vitro transcription of sgRNA
l Goal：synthesize specific RNA molecules for the following experiment
l Material
a. Reagent
DNA model 2μL
T7 transcription buffer 40μL
T7 transcription enzyme mix 4μL
RNase-free H2O 34μL
EDTA solution 500mM
b. Apparatus
pipette
test tubes
PCR
PCR tubes
fridge
l Procedures
1. Add 2μL DNA model, 40μL T7 transcription buffer, 4 μL T7 transcription enzyme mix and 34μL Rnase-free H2O to configurate reaction system.
2. Transport the solution to PCR tubes and put tubes into PCR Amplifier. Keep temperature at 37 degree for 2 hours.
3. Take out the RNA and add 500mM EDTA solution to inactive T7 RNA polymerase.
4. Get the RNA and preserve them in -80°C fridge.
4. Detection of sgRNA cleavage by agarose gel electrophoresis
l Material
a. Reagent
dye 2μL
FEM sample
GBC sample
marker
b. Apparatus
pipette
PCR tubes
Agarose Gel Electrophoresis
l Procedures
1. Inject 2μL of the dye to the FEM and GBC sample in the PCR tubes and then add 1μL to the control sample.
2. Add the mixed solution into the glue with the sequence of marker, FEM, control, GBC and control.
3. Start the electrophoresis.
5. Preparation of LB solution
l Objective
To prepare the basic material for bacteria cultivation
l Material
Reagent
Tryptone 2μL
Yeast Extract
NaCl
Water
Apparatus
conical flask
PCR tubes
Agarose Gel Electrophoresis
Pipette
Weighing paper
Electronic balance
l Procedure
1. Weigh 10g of Tryptone, 5g Yeast Extract and 10g of NaCl and put them in a 1L conical flask.
2. Add about 800 ml of deionized water.
3. Add deionized water to set the volume to 1L.
4. After aliquot, autoclave at 121°C for 20 min.
l Procedure of LB solid culture medium
1. Add 1.5 g agar powder to 100 ml of LB solution, shake well.
2. Autoclave at 121°C for 20 min.
3. When LB solution cooled to about 50°C, add the corresponding concentration of antibiotics.
4. Mix well, pour in a sterile Petri dish, and wait for the agar to solidify before use.
6. Transformation of competent cells of E. coli BL21
l Material
Reagent
pUC57-femA
pUC57-gbcA
pET28a-LbCas12a
DH5α chemically competent cell 200μL
Liquid LB medium
Solid LB medium
Apparatus
Petri dish
Shaker
Pipette
Centrifuge tube (1.5 mL)
Incubator
Bacterial culture tube
l procedure
1. Prepare a 42° water bath.
2. Take 200 μL of E.coli DH5a suspension from a -80 °C freezer and thaw on ice.
3. After thawing the competent states, add plasmid DNA (1-10 ng, and the volume is < 10μL) to the competent cell suspension, gently rotate the off-center tube to mix the contents, and take an ice bath for 30 min.
4. Incubate in a water bath at 42°C for 90s and quickly cool on ice for 3min.
5. Add lmL of LB liquid medium (without antibiotics) to the tube, mix well and culture lh at 37°C with shaking to recover the bacteria.
6. After shaking the above bacterial solution, take 100μL on the screening plate containing antibiotics, place the front side up for 30min, after the bacterial solution is completely absorbed by the medium, put the dish upside down at 37 °C for 16h-24h.
7. IPTG formulation + induction overnight
Material
Reagent
IPTG
Apparatus
Pipette
Centrifuge tube (1.5 mL)
Incubator
Bacterial culture tube
l procedure
1. Weigh 0.238g IPTG
2. Add deionized water to 10 mL and dissolve completely.
3. IPTG solution was sterilized through membrane with a final concentration of 100mM.
4. Induction concentration: 0.01 mM IPTG, 16 ℃ 180 rpm low temperature induction for 12 h overnight
Reconnection
1. Turn 30mL LB liquid medium to six bacterial culture tubes with 5mL medium per tube.
2. Select two bacterial colonies on each of the three LB solid media with transformed E. coli on them (pGEX-6P-1/ pGEX -6P-1-VP1/ pGEX-6P-1-VP1-LTB) and transfer them to the six tubes with one pipette (range: 0.5-2.5μL).
3. Add 50μL ampicillin to each tube with another pipette (range: 20-200μL).
4. Incubate the three tubes in the shaker for 5 hours.
IPTG induction
1. Mark three tubes as the control group and the others as the experimental group.
2. Aspirate 10μL bacteria from each tube, and measure OD600 value.
3. When OD600 value reaches 0.2-0.6, add 25μL IPTG to the three experimental tubes.
4. Incubate the six tubes in the shaker for 3 hours.
8. SDS-PAGE
l Material
|
Reagent |
Volume |
|
ddH2O |
-- |
|
Loading buffer |
-- |
|
Running buffer |
-- |
|
Coomassie brilliant blue staining solution |
-- |
|
Marker |
10μL |
|
SDS-PAGE gel |
1 piece |
|
Apparatus |
Amount |
|
Pipette |
1 |
|
Microcentrifuge tube(1.5mL) |
1 |
|
Electrophoresis chamber |
1 |
|
Box |
1 |
|
Microwave oven |
1 |
l Procedure
Electrophoresis
1. Transfer bacteria liquid to microcentrifuge tubes.
2. Centrifuge the tubes and discard the supernatant.
3. Add ddH2O and loading buffer to the tube.
4. Place the SDS-PAGE gel into electrophoresis chamber and add running buffer to it.
5. Add marker and samples to the hole of the gel
6. Start running.
Staining
1. Clean the gel deionized water 3 times after running.
2. Put the gel into an appropriate amount of Coomassie Brilliant Blue dyeing solution, microwave until close to boiling or just boiling, and stop heating immediately.
3. Then shake on a room temperature shaker for 30 minutes under the condition that the temperature of the dyeing solution is high.
4. Pour out the staining solution.
5. Add an appropriate amount of decolorizing solution to ensure that the dyeing solution can fully cover the gel.
6. Heat the microwave oven until it is close to boiling or just boiling and stop heating immediately.
7. Then shake on the shaker for 30min under the condition that the temperature of the decolorization solution is high. Relatively clear bands of protein are usually observed at this point.
8. Change the fresh decolorization solution, decolorize for 2h, until the blue background is basically removed.
9. Take photos and take samples.
9. Cas12a-crRNA complex in assembly:
Material
Reagent
Cas12a
50nM sgRNA
NEBuffer2.1
Apparatus
Pipette
PCR tube
PCR amplifier
l Procedure
1. 50nM Cas12a + 50nM sgRNA + NEBuffer2.1
2. Incubate in a 20μL system at 37°C for 10min. According to the concentration calculation, the above is added to the PCR tube, expanding to the 50ul system.
3. Centrifuge the mixture for 30 sec.
4. Incubate the solution in a 37°C gradient thermal cycler for 10 min.
5. Add about 100ng of oligo DNA fragments to be cut to the Cas12a-crRNA complex, incubate at 37 °C for 2 hours, and inactivate the protein at 95 °C for 5min.
6. This is followed by 2.5-3% agarose gum identification: sgRNA, oligo DNA, oligo DNA(after cutting)
10.Detection of sgRNA efficiency by ssDNA fluorescent probes
Material
Reagent
Cas12a
50nM sgRNA
NEBuffer2.1
Apparatus
Pipette
PCR tube
PCR amplifier
l Procedure
1. 50nM Cas12a + 50nM crRNA + NEBuffer2.1
2. Incubate in a 20μL system at 37°C for 10min
3. Configure Cas12A-crRNA complex negative control (no incubation required):Add 38.5 μl of reaction buffer to the microtube without protein and sgRNA; Add 0.5 μl of the corresponding plasmid and 3 μl of ssDNA fluorescent probe
4. In each centrifuge tube, add the corresponding plasmid and ssDNA as recorded in the table above.
5. Denature the Cas12a protein by placing the centrifuge tubes in a gradient thermal cycler and incubating at 37°C for 2 h and then at 95°C for 5 min.
6. Measure the ssDNA fluorescence value
11. Bacterial lysis
Material
Reagent
Lysis buffer
Apparatus
Pipette
centrifuge tubes
l Procedure
1. Aspirate 1 ml of bacterial solution into a centrifuge tube and centrifuge at 8,000 rpm for 1 min. Discard the supernatant.
2. Add 50 μL of bacterial lysis buffer to all centrifuge tubes.
3. Place all tubes in a water bath and heat at 80°C for 10 min.
4. Centrifuge all tubes again at 8,000 rpm for 2 min.
5. Measure the concentration and dilute to different concentrations of bacterial liquid.
DETECER tests the sensitivity of different concentrations of bacterial solutions.
12. Bacteria collection
Material
Apparatus
Pipette
centrifuge tubes
l Procedure
1. The whole bacteria are centrifuged at 4 degree with 5000g X 5mins (Balance: 2 tubes with 59.8g; 2 tubes with 62.2 g)
2. Remove the supernatant and retain pellet.
3. Add 10ml resuspension solution, make sure the pellets were dissolved and fell off from the test tube wall.
4. The pellet was sonicated for 20mins at 4 degrees with 300w, 10s/10s.
5. centrifuged it again at 4 degrees, 16000g X 25mins.
6. The rest of the supernatant was transferred into a whole new clean tube and was kept in the ice to maintain the temperature of 4 degree.
12. Ni-chelating affinity purification
Material
Reagent
his-tag purification resin
non-denaturing lysate
affinity column
non denatured washing solution
Apparatus
Pipette
centrifuge tubes
1. Add 1ml 50% his-tag purification resin (mixed well) into the supernatant.
2. centrifuged at 4 degree (1000g X 10s), Ni beads turns from blue into yellow.
3. Add 0.5ml non-denaturing lysate and shake it in ice (4 degree) for 2 hours
4. Transfer the solution into the affinity column.
5. 1ml non denatured washing solution is added to wash the non-relevant materials off. This process was repeated three times.
6. 0.5 ml non-denatured elution solution is added to get our target protein. This process was repeated for four times so we get 4 samples.