| CJUH-JLU-China - iGEM 2024

Reagents Preparation

1. LB liquid medium

2. SOC medium

3. Glyceric CaCl₂ solution

LB (Luria-Bertani) liquid medium

Equipment

Magnetic stirrer

Autoclave sterilizer

Reagents

Take 1L as an example:

Reagent	Amount
Tryptone	10 g
NaCl	5 g
Yeast extract	5 g

Procedure

1. Add the above reagents to a graduated container. Then, add RO water until the total volume reaches 1 liter.

2. Stir them at 660r/min on a magnetic stirrer, until the solutes are completely dissolved.

3. Aliquot the liquid into several conical flasks, 200ml each.

4. Sterilize the medium in an autoclave sterilizer for 20 min at a pressure of 15 psi (1.05 kg/cm2).

Note

1.5g Agar powder was added to obtain LB solid medium.

SOC medium

Equipment

Magnetic stirrer

Autoclave sterilizer

Reagents

Take 100mL as an example:

Reagent	Amount
Tryptone	2 g
Yeast extract	0.5 g
D-Glucose	0.036g
10X inorganic salt solution	1ml

P.S. Reagents of 100ml 10X inorganic salt solution

Reagent	Amount
10mM NaCl	5.85g
2.5mM KCl	1.875g
10mM MgCl₂	9.5g
10mM MgSO4	12g

Procedure

1. Add the above reagents to a graduated container. Then, add RO water until the total volume reaches 100 ml.

2. Stir the mixture at 660r/min on a magnetic stirrer until the solutes are dissolved.

3. Transfer the liquid into a 250ml conical flask.

4. Sterilize the medium in an autoclave sterilizer for 20 min at a pressure of 15 psi (1.05 kg/cm2).

Glyceric CaCl₂ solution

Equipment

Autoclave sterilizer

Reagents

Take 100mL as an example

Reagent	Amount
CaCl₂	0.882 g
Glycerol	10ml

Procedure

1. Add the reagents to a 250ml conical flask, and add RO water until the total volume reaches 100ml.

2. Gently shake the conical disk with hand, until the solutes are completely dissolved.

3. Sterilize the medium in an autoclave sterilizer for 20 min at a pressure of 15 psi (1.05 kg/cm2).

4. 50X TAE

5. 10X TransBuffer

6. 10X Running Buffer

50X TAE

Introduction

50× TAE solution was diluted to obtain 1× TAE solution for gel electrophoresis.

Equipment

Magnetic stirrer

Reagents

Take 1000mL as an example

Reagent	Amount
Tris	242 g
Na₂EDTA•2H₂O	37.2 g
Glacial acetic acid	57.1 ml

Procedure

1. Add the reagents to graduate, and add RO water until the total volume reaches 1L.

2. Stir the mixture on a magnetic stirrer, until the solutes are completely dissolved.

3. Carefully transfer the prepared solution into the specialized bottle labeled for for 50X TAE.

10X TransBuffer

Introduction

10× TransBuffer was diluted to obtain 1× TransBuffer for Western Blot.

Equipment

Magnetic stirrer

Reagents

Take 1L 10X system as an example

Reagent	Amount
Tris	30.2g
Glycine (Gly)	144g

Procedure

1. Add the reagents to a graduate, and add RO water until the total volume reaches 1L.

2. Stir the mixture at 510r/min on a magnetic stirrer, until the solutes are dissolved.

3. Transfer the prepared solution into a specialized bottle labeled for 10X TransBuffer.

10X Running Buffer

Introduction

10× Running Buffer was diluted to obtain 1× Running Buffer for western blot.

Equipment

Magnetic stirrer

Reagents

Take 1L 10X system as an example

Reagent	Amount
Tris	30.2g
Glycine (Gly)	144g
SDS	10g

Procedure

1. Add the reagents to a graduate, and add RO water until the total volume reaches 1L.

2. Stir the mixture at 510r/min on a magnetic stirrer, until the solutes are dissolved.

3. Transfer the liquid into the specialized bottle labeled for 10X Running Buffer.

7. Primary antibody

8. Secondary antibody

9. Stacking gel

Primary antibody

Reagents

Reagent	Amount
TBST	54ml
1% Milk	5ml
GFP tag Rabbit PolyAb	10ul

P.S. Reagents of 1% Milk

Reagent	Amount
5% Milk	1ml
TBST	4ml

Procedure

1. Add 4ml TBST to 1ml 5% Milk to prepare 5 ml of 1% Milk.

2. Thaw the GFP tag Rabbit PolyAb on ice in advance. Take 10 microliters of the antibody and add it to 5 milliliters of 1% milk.

3. Mix the reagents and keep the mixture on ice.

Secondary antibody

Reagents

Reagent	Amount
TBST	90ml
1% Milk	10ml
Goat Anti-Rabbit IgG H&L/HRP	5ul

P.S. Reagents of 1% Milk

Reagent	Amount
5% Milk	2ml
TBST	8ml

Procedure

1. Add 8ml of TBST to 2 ml of 5% Milk to prepare 10 ml of 1% Milk.

2. Thaw the Goat Anti-Rabbit IgG H&L/HRP on ice in advance. Take 5ul of the antibody and add it to 10ml of 1% milk

3. Mix the reagents and keep the mixture on ice.

Stacking gel

Reagents

(Take 10mL as an example)

Reagent	Amount
dd H₂O	7.35ml
30% Acrylamide mixture	1.3ml
1.5M Tris	1.25ml
10% SDS	100ul
10% APS	100ul
TEMED	10ul

Procedure

Mix the above reagents.

10. 12% separating glue

11. Coomassie Brilliant Blue dye

12. Destaining solution

12% Separating glue

Reagents

Take 10mL as an example

Reagent	Amount
dd H₂O	3.3ml
30% Acrylamide mixture	4ml
1.5M Tris	2.5ml
10% SDS	0.1ml
10% APS	0.1ml
TEMED	0.004ml

Procedure

Mix the above reagents.

Coomassie brilliant Blue dye

Reagents

Take 100ml system as an example

Reagent	Amount
Methanol	45ml
RO water	45ml
Glacial Acetic Acid	10ml
Coomassie	0.25g

Procedure

Add the reagents to the specialized bottle labeled for Coomassie brilliant Blue dye, and mix well.

Destaining solution

Reagents

Take 1L system as an example

Reagent	Amount
Ethanol	50ml
Glacial Acetic Acid	100ml
RO water	850ml

Procedure

Add the reagents to the specialized bottle labeled for destaining solution, and mix well.

Bacterial Lab

1. Preparation of competent cells

2. Bacterial Transformation

3. Restriction Digest of Plasmid DNA

Competent cells

Introduction

This protocol is cited from Addgene: Protocol - Competent cells

Equipment

37 °C Shaking incubator

Ice-filled bucket

Centrifuge

Autoclave sterilizer

Four sterilized 50 ml centrifuge tubes, several sterilized 1.5 ml EP tubes (at least 50), one sterilized test tube.

One box of sterilized 1000ul tips, one box of sterilized 200ul tips, more than 3 sterilized 5 ml large tips .

Pipette & tips

Reagents

Monoclonal strains.

One bottle of sterilized non-resistant LB medium (200ml).

Sterilized CaCl₂/Glycerol (50-100 mM CaCl₂, 10-15% glycerol) 100. ml

Procedure

I .Preparation of materials required for the preparation of receptor cells.

1.Add anhydrous calcium chloride (0.88g in 100ml) to the conical flask, add glycerol (10ml in 100ml). Seal and shake well to dissolve. Perform autoclave sterilization.

2.On the first night, add 5 ml of the LB medium with the appropriate resistance system and the corresponding bacterial solution to a 15 ml tube. Place the tube in a slanted position with a half-loose lid and incubate it overnight (16-20 hours) at 37°C in a shaker incubator.

3.Pre-cool the sterilized 50 ml centrifuge tube, 1.5 ml EP tube and 5 ml, 1 ml and 200 μL tips in a -20°C refrigerator; pre-cool CaCl₂/glycerol at 4°C.

II. Preparation of competent cells

1.Add 2 ml of the overnight culture to each 200 ml LB flask. Shake in an incubator at 37°C for 2-3 hours (250 rpm). Grow the cultures until OD600 = 0.375.

2.Dispense four 50 ml tubes for each conical flask, seal them with sealing film and place them on ice for 5-10 minutes. Centrifuge the cells at 1600 g for 10 minutes .

3.Discard the supernatant, add 10 ml of glycerol/CaCl₂ mixture, and centrifuge (1100 g, 4°C, 5 min).

4.Discard the supernatant, add 10ml of glycerol/CaCl₂ mixture, and put it on ice for 30min. Then centrifuge (1100g, 4 ℃, 5min) and discard the supernatant.

5.Dissolve the precipitation with 2ml of glycerol/CaCl2 in total, dispense the solution into EP tubes, 40ul per tube. Then freeze the solution in -80 ℃ refrigerator .

Bacterial Transformation

Introduction

This protocol is cited from Addgene: Protocol - Bacterial Transformation

Equipment

37 °C Shaking incubator

37 °C Stationary incubator

Water bath at 42 °C

Ice-filled bucket

Microcentrifuge tubes

Sterile spreading device

Reagents

LB agar plate (with appropriate antibiotic)

SOC media

Competent cells

Plasmids to transform

Procedure

1. Take competent cells out of -80°C and thaw them on the ice.

2. Remove agar plates (containing the appropriate antibiotic) from storage at 4°C and let them warm up to room temperature and then (optional) incubate them in 37°C incubator.

3. Mix 2μl of plasmids into 50 μL of competent cells in a microcentrifuge tube. Gently mix them.

4. Incubate the competent cell/plasmid mixture on ice for 20-30 mins.

5. Heat shock each transformation tube by placing the bottom half of the tube into a 42°C water bath for 90 seconds.

6. Put the tubes back on ice for 2 minutes.

7. Add 500μl of LB or SOC medium (without antibiotic) to the bacteria and grow them in a 37°C shaking incubator for 1-1.5 hours.

8. Plate the transformation (about 150-200μL ) onto a LB agar plate containing the appropriate antibiotic.

9. Incubate the plates at 37°C overnight.

Restriction Digest of Plasmid DNA

Introduction

This protocol is cited from Addgene: Molecular Biology Protocol - Restriction Digest of Plasmid DNA

Equipment

Pipette & tips

Reagents

Aliquot of plasmid DNA solution

Appropriate restriction enzyme

dd H₂O

rcutsmart

Procedure

1. Select restriction enzymes to digest plasmids:

We use EcoR I, EcoR V, Hpa I, Mlu I, Xba I, Nhe I, Hind III.

The amount of restriction enzyme is 1μL per 20μL system.

(Detailed enzyme information followed https://nebcloner.neb.com/#!/redigest )

2. Calculate the system of restriction digest(20ul)

P.S: Usually, dd H₂O is added to reach the total volume.

3. Use a pipette to add the reagents in order

Usually, we follow the order: ddH₂O, plasmids, rcutsmart, restriction enzyme which must be added last.

4. Digest under specific temperature and duration

Usually, we digest at room temperature overnight.

5. Agarose Gel Electrophoresis for verification

(Detailed operation could follow the protocol of Agarose Gel Electrophoresis)

4. Agarose gel electrophoresis (DNA)

5. Agarose gel electrophoresis (RNA)

6. PCR

Agarose Gel Electrophoresis (DNA)

Introduction

This protocol is cited from Addgene: Protocol - How to Run an Agarose Gel

Equipment

Ice bags

Casting tray

Well combs

Voltage source

Gel box

UV light source

Microwave

Reagents

TAE (Detailed information followed protocol of TAE)

Agarose

Nucleic acid dye

Procedure

1. Measure the required amount of agarose.

2. Mix 1 g of agarose powder with 120 ml of 1xTAE buffer in a microwavable flask.

3. Microwave the mixture for 1 to 3 minutes until the agarose is completely dissolved.

4. Let agarose solution cool down to approximately 50 °C.

5. Add the nucleic acid dye, usually about 20μl per 160 ml of gel.

6. Pour slowly to avoid bubbles which will disrupt the gel. Any bubbles can be pushed away from the well comb or towards the sides/edges of the gel with a pipette tip.

7. Place the newly poured gel at room temperature for 30 to 45 minutes until it has completely solidified.

8. Add loading buffer to each of your DNA samples.

9. Once solidified, place the agarose gel into the gel box (electrophoresis unit).

10. Fill the gel box with 1xTAE buffer until the gel is covered.

11. Carefully load a molecular weight ladder into the first lane of the gel.

12. Carefully load your samples into the additional wells of the gel.

13. Run the gel at 135V until the dye line is approximately 75-80% of the way down the gel. The typical run time is about 45 minutes, which may vary depending on the gel concentration and voltage.

14. Turn OFF the power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.

15. Using any device with UV light to visualize your DNA fragments. The DNA fragments are usually referred to as ‘bands' due to their appearance on the gel.

Agarose Gel Electrophoresis (RNA)

Introduction

This protocol is cited from NorthernMax™ Kit USER GUIDE

Equipment

Ice bags

Casting tray

Well combs

Voltage source

Gel box

UV light source

Microwave

Polypropylene microcentrifuge tubes,

Reagents

Formaldehyde Load Dye

Agarose-LE

10X Denaturing Gel Buffer

10X MOPS Gel Running Buffer

Nucleic acid dye

RNaseZap™ RNase Decontamination Solution

SYBR^® Gold Nucleic Acid Gel Stain

Procedure

I. Prepare gel

1. Melt 1 gm agarose in 82 mL RNase-free water for every 100 mL of gel.

2. Melt in a microwave oven, with frequent agitation, until the agarose is completely in solution.

3. Add 18mL 10X Denaturing Gel Buffer per 100 mL of gel and mix well.

4. Pour the gel to about 0.6cmin thickness and add 10μL 10000X SYBR^® Gold Nucleic Acid Gel Stain .mix well by pipette tip gently; try to avoid forming bubbles in the gel solution.

a) Because of the formaldehyde, the gel should be poured in a fume hood; once the agarose is solidified, it can be removed from the hood.

b) Bubbles should be eliminated by popping them with a heated glass or metal rod, or by pushing them to the edges of the gel with a clean pipet tip.

c) The comb should be positioned ~1 cm from the top of the gel, at a height of ~2 mm. To increase well capacity, use combs with thicker teeth rather than pouring a thicker gel.

5. Allow the gel to solidify at RT remove the comb.

a) After the gel has solidified, carefully remove the comb; a thin layer of 1X Gel Running Buffer poured over the gel surface before removing the comb may help to prevent the wells from tearing when the comb is removed.

6. Set up the electrophoresis chamber.

Do not let gels sit in the buffer for more than ~1 hour before loading.

a) Position the gel tray in the electrophoresis chamber with the wells next to the cathode (negative/black) lead.

b) Dilute the 10X MOPS Gel Running Buffer to 1X with nuclease-free water and cover the gel with about 0.5–1 cm running buffer.

c) STOPPING POINT Gels can be wrapped in plastic and stored in the refrigerator overnight.

II. Prepare RNA

1. Mix sample RNA with 3 volumes NorthernMax™ Formaldehyde Load Dye and one tenth volumes 10X SYBR^® Gold Nucleic Acid Gel Stain .

a) Up to 30 μg total RNA or poly (A+) RNA can be loaded per lane. Using more than 30 μg of total RNA overloads the gel, and/or exceeds the RNA binding capacity of the membrane. If molecular weight markers are used, they should also be mixed with load dye at this step. If the total volume exceeds the capacity of the wells, the RNA must be precipitated and resuspended in a smaller volume of RNase-free water or dissolved directly in ~20 μL NorthernMax™ Formaldehyde Load Dye

2. Incubate the samples 15 min at 65°C.

a) This incubation denatures RNA secondary structure. A dry heat block is recommended for this step to avoid potential contamination of the samples with water from a water bath.

3. Centrifuge briefly to collect the contents, then place on ice.

III. Run the gel

1. Load the RNA samples into the wells of the gel.

a) Use RNase-free pipette tips. To keep the samples as dense as possible, make sure there is no air trapped in the end of the pipette tip. Place the tip just inside the top of the well, expel the sample slowly, then gently raise the pipette tip out of the well.

2. Run the gel at 110v,45min.

Polymerase Chain reaction (PCR)

Introduction

This protocol is cited from Addgene: What is Polymerase Chain Reaction (PCR)

Equipment

Thin-walled PCR tube

Ice-filled Bucket

PCR Machine

Reagents

Template DNA

10X Taq buffer with MgCl₂

dNTP mix

Reverse Primer

Sterile ddH₂O

Taq DNA Polymerase

Agarose Gel

Procedure

1. Place thin-walled PCR tubes on the ice

2. Set up a reaction system (Keep all your reagents on ice):

a) 35 μl of 5X Prime buffer with MgCl₂

b) 14 μl of dNTP mix

c) 21 μL of Forward Primer

d) 21 μL of Reverse Primer

e) 75.25 μL of ddH₂O

f) 1.75 μL of Taq DNA Polymerase

3. Place reaction tubes in the PCR machine.

4. Set annealing temperature 5°C below the primer melting temperature (Tm).

5. Set extension step at 1-2 minutes per kilobase of product depending on whether you are using a polymerase with proofreading capabilities.

6. Initial Denature for 1 minute at 98°C.

7. Denature for 10 seconds at 98°C.

8. Anneal primers for 5 seconds at 55°C

9. Extend DNA for 1.5 minutes at 72°C.

10. Repeat steps 7-9 for 30 cycles.

11. Final Extension for 2 minutes at 72°C.

12. Run 2 μL on a gel to check size and concentration of PCR product.(Details could follow the protocol of Agarose Gel Electrophoresis)

7. Plasmid Maxiprep

8. Plasmid Miniprep

9. RNA extraction

Plasmid Maxiprep

Vigorous Plasmid Maxiprep

Introduction

This protocol is cited from plasmid.pdf (vigorousbiol.com)

MaxPrep is the name of a technique to isolate plasmid DNA from E. coli containing plasmid of interest by alkaline lysis method .

Equipment

Ice-filled bucket

Centrifuge

BeyoVortex™ Basic Vortex Mixer

Reagents

Buffer I (resuspension )

Buffer II (lysis)

Buffer III (neutralization )

Buffer IV(impurity removal solution A)

Buffer V (impurity removal solution B)

Buffer VI (impurity removal solution C)

RNase A

70% ethanol (14mL 75% ethanol +1mL ddH₂O)

Isopropanol

ddH₂O

Procedure

I.Buffer preparation

1. Add the provided RNase A solution to Buffer I (use 1 vial RNase A per bottle Buffer I), mix, and store at 2-8°C.

II.Harvest bacterial precipitate

1. Take bacteria solution from the overnight culture (200mL LB in a conical flask with appropriate antibiotic) and transfer it into a 50 mL centrifuge tube.

2. Centrifuge it at 5000 rpm for 10 minutes at 4℃ to get the bacterial precipitate. Try to remove all traces of supernatant, use clean paper tissue to absorb the fluids inside the tube wall.

III.Extraction of plasmid

1. Add 5 mL of Buffer l to the bacterial centrifuge tubes, mix and shake the bacterial precipitate well to disperse it completely until no flocs exist. Transfer the bacterial suspension into a 50 mL centrifuge tube.

2. Add 5 mL of Buffer ll, gently invert the centrifuge tube 6-8 times, and leave it at room temperature for 5 min, so that the bacteria are completely lysed and the solution is clear.

3. Add 5 mL of Buffer lll, immediately invert the centrifuge tube 6-8 times and mix well until white flocculent is produced. Leave on ice for 12 min.

4. Centrifuge the above lysate at 12,000xg for 15 min at 4°C. Carefully aspirate the supernatant and transfer to a new 50 mL centrifuge tube

5. Add 10 mL of isopropanol. Mix well by inversion and leave on ice for 12 min.

6. Centrifuge at 12,000xg for 10 min at 4°C. Carefully discard the supernatant, invert and gently drain off the residouble liquid, add 0.5 mL of Buffier l to completely dissolve the precipitate clumps.

7. Transfer to a new 1.5mL centrifuge tube and leave at room temperature for 12min.

8. Centrifuge the crude extract of plasmid at high speed for 2min at room temperature in a desktop centrifuge, and transfer the supernatant into a new 1.5mL centrifuge tube.

IV.Plasmid purification

1. Add 100μl of Buffer IV (impurity removal solution A) to 0.5mL of plasmid crude extract, gently mixi, 2,000xg centrifugation for 2min, the supernatant should be transferred to a new centrifuge tube.

2. Add 100μl of Buffer IV (Impurity Removal Solution A), mix gently, centrifuge at 12,000xg for 5min and transfer the supernatant to a new tube.

3. Add 70μL of BufferV (Impurity Removal Solution B), mix gently, centrifuge at 12,000xg for 5 min, transfer the supernatant to a new centrifuge tube.

4. 0.5mL of isopropanol should be added, mixed well, and left at room temperature for 10 min. 12,000xg centrifugation at room temperature for 10min, the supernatant should be discarded, washed gently with 1mL of 70% ethanol, the liquid should be discarded, and invert the tube. Cool it for 5 min at room temperature.

5. Dissolve precipitate with 0.5mL of ddH₂O

6. Add 200ul of Buffer VI (Impurity Removal Solution C), mix well and leave on ice for 20 min, centrifuge at 12,000xg for 10min at room temperature. Discard the supernatant, gently add 1ml of 70% ethanol to wash twice, invert and cool at room temperature for 5-10 min to make the ethanol evaporate completely.

7. Add appropriate amount of dd H₂O (250μL) to dissolve the precipitate (can be shaken in a 37°C water bath to aid dissolution)

TIANGEN Plasmid Maxiprep

Introduction

This protocol is cited from EndoFree Maxi Plasmid Kit

EndoFree Maxi Plasmid Kit is based on alkaline lysis technology followed by adsorption of DNA onto silica membrane in the presence of high salt, and uses Buffer P4 and Filtration CS1 to effectively wipe off the endotoxin and protein impurity.

Equipment

Ice-filled bucket

Centrifuge

BeyoVortex™ Basic Vortex Mixer

Filtration CS1

Spin Columns CP6

50 ml Collection Tubes

Reagents

LB agar plate (with appropriate antibiotic)

Buffer BL

Buffer P1

Buffer P2

Buffer P4

Buffer PW

Buffer TB

96-100% ethanol

Isopropanol

RNase A

Procedure

I.Buffer preparation

1. Add the provided RNase A solution to Buffer P1 (use 1 vial RNase A per bottle Buffer P1), mix, and store at 2-8°C.

2. Add ethanol (96-100%) to Buffer PW before use,

3. Check Buffer BL, P2 and P4 before use for salt precipitation. If necessary, dissolve the buffer by warming at 37°C.

4. Avoid direct contact between Buffer P2 and P4, immediately close the lid after use.

II.Harvest bacterial precipitate

1. Take bacteria solution from the overnight culture (200mL LB in a conical flask with appropriate antibiotic) and transfer it into a 50 mL centrifuge tube.

2. Centrifuge it at 5000 rpm for 10 minutes at 4℃ to get the bacterial precipitate. Try to remove all traces of supernatant, use clean paper tissue to absorb the fluids inside the tube wall.

III.Plasmid extraction

1. Resuspend pelleted bacterial cells in 8 ml Buffer P1. The bacteria should be resuspended completely by vortex or pipetting up and down until no cell clumps remain.

2. Add 8 ml of Buffer P2 and mix thoroughly by inverting the tube 6-8 times, then incubate at room temperature for 5 min.

3. Add 8 ml of Buffer P4, and mix immediately and thoroughly by gently inverting 6-8 times, until the whole solution become cloudy. Incubate at room temperature for 10 min. Centrifuge for 5-10 min at 8,000 rpm, the white material should be in the bottom of the centrifuge tube. Transfer the supernatant into a Filtration CS1. Gently insert the plunger into the Filtration CS1 and filter the cell lysate into a new 50 ml tube.

4. Column equilibration: place a Spin Column CP6 into a 50 ml Collection Tube and add 2.5 ml of Buffer BL to Spin Column CP6. Centrifuge for 2 min at 8,000 rpm. Discard the flow- through, and place Spin Column CP6 into the same Collection Tube

5. Add 0.3 times the volume of isopropanol to the cleared lysate, mix completely by reverting upside and down and then transfer all solution to the Spin Column CP6.

6. Centrifuge for 2 min at 8,000 rpm. Discard the flow-through and place the Spin Column CP6 back into the same Collection Tube.

7. Add 10 ml of Buffer PW to the Spin Column CP6 and centrifuge at 8,000 rpm for 2 min. Discard the flow-through and place the Spin Column CP6 back into the same Collection Tube.

8. Repeat step 9.

9. Add 3 ml of 100% ethanol to the Spin Column CP6 (put the CP6 in a Collection Tube). Centrifuge for 2 min at 8,000 rpm, discard the flow-through.

10. Put Spin Column CP6 back to Collection Tube, centrifuge at 8,000 rpm for 5 min for removing residual ethanol.

11. To elute DNA, place the Spin Column CP6 in a clean 50 ml Collection Tube and add 1-2 ml of Buffer TB to the center of the membrane and incubate 5 min at room temperature, centrifuge at 8,000 rpm for 2 min. Transfer the eluate from 50 ml centrifuge tube to a clean 1.5 ml centrifuge tube and put at -20 °C for storage.

TIAN GEN Plasmid Miniprep

Introduction

This protocol is cited from the handbook of TIANprep Rapid Mini Plasmid Kit

Equipment

Spin Columns CP3

2ml Collection Tubes

Centrifuge

Reagents

RNase A (10 mg/ml)

Buffer P1

Buffer P2

Buffer P5

Buffer PWT

TIANRed

Procedure

1. Harvest 1 to 4 ml bacterial cells in a microcentrifuge tube by centrifugation at 12,000 rpm in a conventional, table-top microcentrifuge for 1 minute at room temperature. Then, remove all traces of supernatant by inverting the open centrifuge tube until all medium has been drained.

2. Resuspend the pelleted bacterial cells in 150 μl of Buffer P1 by pipetting or vortexing.

3. Add 150 μl of Buffer P2 and gently mix by inverting the tube 6 to 8 times.

4. Add 350 μl of Buffer P5 and mix immediately and quickly mix by inverting 12 to 20 times. The solution should become cloudy. Centrifuge for 2 minutes at 12,000 rpm in a table-top microcentrifuge.

5. Transfer the supernatant from step 4 to the Spin Column CP3 (Placed in a Collection Tube) by pipetting. Centrifuge for 30 seconds at 12,000 rpm. Discard the flow-through and set the Spin Column CP3 back into the Collection Tube.

6. Wash the Spin Column CP3 by adding 300 µl of Buffer PWT and centrifuge for 30 seconds at 12,000 rpm. Discard the flow-through and put the Spin Column CP3 back into the Collection Tube.

7. Centrifuge for an additional 1 minute at 12,000 rpm to remove residual wash buffer.

8. Place the Spin Column CP3 in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 to 100 μl of ddH₂O to the center of the Spin Column CP3, centrifuge for 30 seconds at 12,000 rpm.

RNA Extraction

Introduction

This protocol is cited from EasyPure® RNA Kit

Equipment

Ice-filled bucket

Eppendorf Microcentrifuges

miRNA/RNA Spin Columns with Collection Tubes

RNase-free Tube(1.5mL)

Reagents

WashBuffer10(WB10, Add 48 mL of absolute ethanol toWB10 prior to use)

RNase-free Water

Absolute ethanol.

Procedure

I. DNA template preparation：Single enzyme digestion

1. In order to obtain a specific length RNA, the plasmids must be completely linearized.

2. Digest at 37℃ for 2h

II. In vitro RNA transcription

1. Thawing reagents:

a) Centrifuge the T7 RNA Polymerase Mix briefly and place on ice. Thaw 10× Transcription Buffer and ribonucleotides (ATP, CTP, GTP, UTP), mix and centrifuge to the bottom of the tube, place 10× Transcription Buffer at room temperature, and place 4 types of ribonucleotides on ice.

2. Assembly transcription reaction at room temperature

Prepare the reaction system according to the following system:

Reagents	Amount
RNase free H₂O	Up to 20 μL
10×Transcription buffer	2 μL
CTP/GTP/ATP/UTP(100 mM each)	2 μL each
Template DNA	1 μg
T7 RNA Polymera Mix	2 μL
RNAase Inhibitor	0.5 μL

Note:

1. The reaction is configured at room temperature. Since 10× Transcription Buffer contains spermidine, the concentration of spermidine too high will cause DNA template precipitation at low temperature.

2. Short transcript (<100 nt), 2 µg template can be used, transcription time increased to 4-8 hs.

3. Perform the reaction in a PCR machine with the hot lid open to prevent the reaction solution from evaporating for a long time.

4. The reaction product may have a white precipitate.

5. To ensure the reagents and containers must without RNase contamination, we add 0.5ul RNase Inhibiter into each system.

6. Incubate at 37°C for 4 hours

7. Before purification, add 80μL RNase free H2O to dilute the product to 100 μL.

10. RNA purification

11. Strain preservation

12. Ultrasonic disruption of bacteria

RNA purification

Introduction

This protocol is cited from EasyPure® RNA Kit

Equipment

Ice-filled bucket

Eppendorf Microcentrifuges

miRNA/RNA Spin Columns with Collection Tubes

RNase-free Tube(1.5mL)

Reagents

WashBuffer10(WB10, Add 48 mL of absolute ethanol toWB10 prior to use)

RNase-free Water

Absolute ethanol.

Procedure

I .Purification

1. Transfer it to a clean 1.5 mL RNase-free centrifuge tube. Add 1.25 volumes (125μL)of absolute ethanol to the tube .Some precipitates may appear at this moment. Invert the tube gently to mix well.

2. Add all the solution and precipitates into the miRNA Spin Column. Centrifuge at 12,000xg for 30 seconds at room temperature, and discard the flow-through (If the volume of the mixture is larger than the maximum sample volume the column can process, repeat this step until all the mixture has been loaded)

3. Add 500 ul of WB10 (check to make sure that absolute ethanol has been added prior to use) into the spin column. Centrifuge at 12,000xg for 30 seconds at room temperature. Discard the flow-through.

4. Repeat step (3) once.

5. Centrifuge at 12,000 xg for 2 minutes at room temperature to remove ethanol residue thoroughly.

6. Place the miRNA spin column into a 1.5 mL RNase-free tube. Add 30 ul of RNase-free Water to the center of the spin column matrix and incubate at room temperature for 1 minute.

7. Centrifuge at 12,000xg for 1 minute to elute miRNA.

8. Store the isolated miRNA at -80°C

II. RNA quantification

a) Detect the concentration of RNA.

III. RNA size and quality detection

1. In order to determine the size, integrity and quality of RNA, agarose gel electrophoresis or polyacrylamide gel electrophoresis is required for detection.

2. Agarose electrophoresis （Detailed operation could follow the protocol of Agarose Gel RNA Electrophoresis）

Strain preservation

Equipments

Pipette & pipette tips

1.5ml EP tubes

-80°C Refrigerator

Reagents

Glycerol

Bacterial solution

Procedure

1. Add 200ul of glycerol, and then 800ul of bacterial solution into a 1.5ml EP tube.

2. Thoroughly mix and store the strain in the refrigerator at -80 °C.

Ultrasonic disruption of bacteria

Equipments

Ultrasonic disruptor

Centrifuge

Pipette & pipette tips

50ml centrifuge tubes

Ice-filled glass beaker

5ml EP tubes

Reagents

PBS solution

Bacterial solution

Procedure

1. After centrifugation, 10 ml of PBS solution was added to the bacterial solution and blown to mix well

2. Fix the 50 ml centrifuge tube on an iron stand and immerse the ultrasound probe into the bacterial solution

3. Immerse the 50 ml centrifuge tube in a 250 ml glass beaker filled with ice, and fix the device .

4. Adjust the instrument parameters as follows: power at 180W, ultrasonic working time of 10s, ultrasonic interval time of 40s.

5. Disrupt the bacteria: Repeat 10 times of every 10-second working time with a 40-second interval time, which is regarded as one project. In total, perform four projects.

6. Centrifuge the disrupted bacterial solution at 5000rpm for 10min. Take the supernatant and store it in a 5ml EP tube.

13. Multi-Mode Microplate Reader

14. Coomassie staining of the gel

15. Western Blot

Multi-Mode Microplate Reader

Introduction

This protocol is cited from specification c18a8b7b538cf35b6e2c7850ccf739f8.pdf (allsheng.com)

Equipment

Feyond-A300 Microplate Reader

Reagents

Protein samples

Procedure

1. All protein samples were diluted to a concentration of 1.8μg/μL with a sample volume of 100 ul. Determine the fluorescence intensity at an excitation wavelength of 488 nm and an emission wavelength of 525 nm, with medium to 525 nm and medium-high PMT gain.

2. The remaining operations should be carried out strictly in accordance with the instrument instructions.

Coomassie staining of the gel

Introduction

This protocol is cited from Western blot protocol | abcam

Equipment

12% SDS-PAGE gel

Gel electrophoresis apparatus

Orbital Shaker

Reagents

0.25% Coomassie Blue stain (store at 4℃)

Destaining solution (Detailed information followed protocol of Destaining solution)

Procedure

1. Observe the colored bands (Marker) of the pre-stained molecular weight ladder. Colored bands should be clearly visible on the gel.

2. Stain the SDS-PAGE gel in Coomassie Blue stain. Immerse the gel in the Coomassie stain and hold for 30-60minutes.

3. Wash the gel extensively with water 3 times preliminarily.

4. Wash the gel with destaining solution for 15-30minutes until the background is removed.

Usually, protein bands appear 1-2 hours after decolourisation.

(If not, replace the destaining solution to RO H₂O and continue decolourisation overnight.)

5. Blue bands indicate proteins remaining on the gel. The shade of the bands determines the protein content.

6. After completing the decolourisation, the gel can be stored in water and used for subsequent photographs etc.

Western Blot

Introduction

This protocol is cited from https://www.abcam.com/en-us/technical-resources/protocols/western-blot

Equipment

Ice bags

Gel running apparatus

Centrifuge

Membrane

Transfer apparatus

Transfer cassette

Rolling device

Imaging Systems

Developing cassette

Reagents

Prestained Protein Ladder

Wash buffer(TBST)

Running buffer

Transfer buffer

Blocking buffer(5% milk)

5% stacking gel &12% separating gel

Primary antibody& secondary antibody

Methanol

Developer(ECL solution)

Procedure

I.Sample Preparation

1. Centrifuge the suspension at 5000 rpm several times at 4°C to pellet the thallus.

2. Keep the supernatant in place in a fresh tube on ice.

3. Use ultrasonication to disrupt the bacteria

4. Determine the protein concentration

5. Dilute the aliquots in Sample buffer.

II.Loading and running the gel

1. Select an appropriate SDS-PAGE gel for your protein and set up the running apparatus.

(Normally we use stacking gel and 12% separating gel)

2. Load an equal mass of protein from each sample into the gel.

3. Run the gel according to the protein situation.

Usually, we use 90V for stacking gel and 135v for separating gel

III.Transfer

1. Soak the membrane in methanol, then in transfer buffer.

2. Assemble the SDS-PAGE gel and the membrane in the transfer cassette.

i. According to this order: White board - sponge - two pieces of thick filter paper - one piece of thin filter paper - film --Glue - one thin filter paper - two thick filter paper - sponge - - blackboard - glue - one thin filter paper - two thick filter paper - sponge -Blackboard

ii. Ensure that the gel is closest to the negative electrode and the membrane is closest to the positive electrode.

3. Set up the Transfer apparatus on ice, 200mA current for 2h.

IV.Blocking and antibody incubation

1. Place the membrane in a container and cover with blocking buffer.

(The blocking buffer is 5% milk )

2. Cover the membrane with primary antibody, place it on the table, shaking overnight under 4℃.

3. Wash the membrane three times with TBST, 10 min each.

4. Cover the membrane with secondary antibody, place it on the table, and shake for 1h.

5. Wash the membrane three times with TBST again, 10 min each.

V.Detection

1. Pre-cool the imaging system to -80°C.

2. Add developer to the membrane.

3. Scan membranes in the imaging system.

Cell Culture Lab

1. Cell Culture

2. Transwell

3. Wound healing

Cultured cell lines

Introduction

This protocol is cited from

the-complete-guide-to-cell-culture.pdf (ptgcn.com)

Equipment

Aspiratory pump

Autoclave

Cell counter

Cell culture flasks

Cell culture-grade Petri dishes

Cell culture-grade tubes of various sizes

Cell culture hood

Cell culture microwell plates

Cell incubator

Centrifuge

Fridge/freezer

Pipettes of various capacities

Serological pipettor

Sterile filters

Reagents

70% ethanol antiseptic

Media

Sera

Cell media additives

Procedure(For Adherent cells)

1. Remove medium.

2. Wash cells with calcium- and magnesium-free balanced salt solution (phosphate buffered saline, Hanks' balanced salt solution, etc).

3. Add detaching agent (e.g., trypsin). Incubate at 37°C until cells are fully detached from the dish (2-20 min depending on the cell line).

4. Resuspend cells in fresh medium, pipette thoroughly to obtain single cell suspension. If your medium does not contain serum, you need to inactivate detaching agents, e.g., by addition of trypsin inhibitors. Measure total number of cells (see: counting cells).

5. Plate cells onto a new dish at the desired cell density.

Procedure(For Suspension cells)

1. Collect cells with medium and briefly centrifuge (150-300 xg, 3-5 min) to pellet cells.

2. Remove medium and gently resuspend cells with a balanced salt solution. Briefly centrifuge (150-300 xg, 3-5 min) to pellet cells.

3. Remove salt solution and resuspend cells in fresh medium. Measure total number of cells (see: counting cells).

4. Plate cells onto a new dish at desired cell density.

Transwell

Introduction

This protocol is cited from

Transwell Assay Protocol | Creative Bioarray (creative-bioarray.com)

Equipment

Ice-filled bucket

Microcentrifuge

RNase free microcentrifuge tubes

Thermostatic incubator

Transwell chambers

Microscope

Culture dish

Counting plate

24-well plate

Wet box

Pipette & tips

EP tubes

Reagents

Trypsin digestion solution

Matrigel

DMEM

Serum

OPti-Medium

RNA iMax

DEPC H₂O

Cells

Mimic-142-3p and Mimic-210-3p

Inhibitor-142-3p and Inhibitor-210-3p

Control

Methanol

Giemsa stain

Distilled water

Procedure

I. Prepare the matrigel coating:

1. Matrigel is thawed and liquefied on ice, and then diluted with DMEM(Matrigel:DMEM=1:4).

2. Add 40 µL of matrigel to per well of the upper chamber, and put it in a 37°C incubator for 45 minutes.

II. Digest the cells:

1. Use a pipette to aspirate out the waste liquid in the culture dish under negative pressure, add PBS to rinse and then suck out the liquid again

2. Add trypsin digestion solution

3. Add DMEM, rinse the bottom of the culture dish and collect cells

4. Microcentrifuge cells at 1000rpm for 5min

5. Discard the supernatant, add PBS again, microcentrifuge again and remove the supernatant

6. Add DMEM and mix well

7. Dilute the cells to a reasonable concentration for easy liquid collection, and count them with a counting plate after dilution until a reasonable concentration is reached

III. Prepare the overexpression conditions:

(Taking mimic-142-3p and inhibitor-142-3p as an example)

1. Add OPti-Medium and RNA iMax into three EP tubes , (100μl of OPti-Medium and 1μl of RNA iMax each ),mix well,and hold at room temparature for 5min

2. Dilute mimic-142-3p with DEPC H2O (1OD mimic-142-3p + 125 μl DEPC H2O);

Dilute inhibitor-142-3p with DEPC H2O (1OD inhibitor-142-3p + 125 μl DEPC H2O);

Dilute control with DEPC H2O (0.5OD Control + 125 μl DEPC H2O)

3. Separately add 1ul of mimic-142-3p, 1ul of inhibitor-142-3p, 1ul of control into the aforesaid three EP tubes,mix well,and hold at room temparature for 20min.

Mimic-210-3p and inhibitor-210-3p are followed by the above steps.

IV. Add reagents to upper chamber and lower chamber :

1. After the upper chamber glue has set, add 100ul of cell suspension to per well of it,add 100ul of overexpression conditions to per well of it.

2. Add 650ul of DMEM containing 20% serum to each well of the lower chamber(500ulDMEM+150ul serum)

V. Culture cells:Put the 24-well plate into the thermostatic incubator for 48h.

VI. Giemsa staining:

1. Fix the cells in methanol for 10 min, discard the fixative and rinse the cells with distilled water.

2. The Giemsa stain was diluted with PBS (Giemsa stain:PBS=1:9). Add 850ul of Giemsa stain to each well ,and stain with wet box at 37℃ for 1h or overnight at room temperature.

3. Rinse well with distilled water and place under the microscope.

VII. Invasive cell counting: Randomly select 3-5 fields of view under the microscope and count the number of cells migrating to the lower side of the membrane.

VIII. Data analysis: Based on the counting results, calculate the percentage of invaded cells or related statistics.

Wound healing

Equipment

Microcentrifuge

RNase free microcentrifuge tubes

Thermostatic incubator

Microscope

Culture dish

Counting plate

Six-well plate

Pipette & tips

Reagents

PBS

Trypsin digestion solution

DMEM

Cells

OPti-Medium

RNA iMax

Mimic-142-3p and Mimic-210-3p

Inhibitor-142-3p and Inhibitor-210-3p

Control

DEPC H₂O

Procedure

I. Take a six-well plate and mark three parallel lines on the bottom. Label the corners with information about each well.

II. Digest the cells:

1. Use a pipette to aspirate out the waste liquid in the culture dish under negative pressure. Add PBS to rinse and then suck out the liquid again

2. Add trypsin digestion solution

3. Add DMEM, rinse the bottom of the culture dish and collect cells

4. Microcentrifuge cells at 1000rpm for 5min

5. Discard the supernatant, add PBS again, microcentrifuge again and remove the supernatant

6. Add DMEM and mix well

7. Dilute the cells to a reasonable concentration for easy liquid collection, and count them with a counting plate after dilution until a reasonable concentration is reached

8. Cells were plated in a general cell density of 5*103 cells per well.

III. Prepare the overexpression conditions:

(Taking mimic-142-3p and inhibitor-142-3p as an example)

1. Add OPti-Medium and RNA iMax into three 5mL centrifuge tubes , (950μl of OPti-Medium and 5μl of RNA iMax each ),mix well, and hold at room temperature for 5 minutes.

2. Dilute mimic-142-3p with DEPC H2O (1OD mimic-142-3p + 125 μl DEPC H2O);

Dilute inhibitor-142-3p with DEPC H2O (1OD inhibitor-142-3p + 125 μl DEPC H2O);

Dilute control with DEPC H2O (0.5OD Control + 125 μl DEPC H2O)

3. Separately add 5ul of mimic-142-3p, 5ul of inhibitor-142-3p, 5ul of control into aforesaid three 5mL centrifuge tubes,mix well,and hold at room temparature for 20min

4. Overexpression conditions were added in an amount of 900μL per well.

Mimic-210-3p and inhibitor-210-3p are followed by the above steps.

IV. Put the six-well plate into the thermostatic incubator to culture cells. In conjunction with the actual overexpression time required, when the cells are full grown, the six-well plate is withdrawn and three parallel lines are drawn with the tips along the previously drawn straight line.

V. Wash slowly with PBS and add 3mL of serum-free medium. Select the spot for photographing with an inverted microscope, and mark it as 0h. Then capture images at 24h and 48h.