Connection

1. ABSTRACT

This protocol is used to connect two or more pieces of DNA together

1. PREPARATION

Set up a small box with ice, put DNA and enzymes on it. Prepare metal bath was heated to 50 °C and used for Gibson assembly

• 1. Select the appropriate connection method according to the experimental Situation. This protocol is based on Seamless Cloning Kit by Beyotime. This protocol is used to connect two pieces of DNA.
• 2. Select an appropriate cloning site on the vector that will be linearized.
• 3. Vector linearization: Primers with homologous sequences were designed on Snap Gene software and the sequences were sent to Engine Biotech for synthesis PCR of the inserts DNA fragments.
• 4. Amplify the insert DNA fragments with homologous sequences (for homologous recombination) of vector-upstream or -downstream by PCR using high fidelity DNA polymerase.
• 5. Detect DNA concentration of linearized vectors and inserts by Nanodrop.
• 6. Dilute linearized vectors and inserts before recombination to make sure the loading accuracy. The volume of each component loaded should be no less than 1 μL.
• 7. Calculation of the amount of vectors: The molar ratio of each insert to vector was 3:1 (10-100 ng of purified PCR fragment, 50-100 ng of linearized vector), 2X Seamless Cloning Mix with 10 µL. The total system was 20 µL, and the remaining volume was replenished by Nuclease free water.

Constituent	Add the ratio / volume
Each insert fragment /The molar ratio of the vector	3:1
Purified PCR fragments	10-100 ng
Linear vector	50-100 ng
2X Seamless Cloning Mix	10 µL
Nuclease free water	add to 20 µL



• 8. The above reaction system was incubated at 50 °C for 15 min (1-2 fragments), 30 min (3 fragments) or 60 min (4-5 fragments). If the reaction cannot be performed immediately after the completion, the reaction sample can be stored at-20 °C.

Gel recovery

1. ABSTRACT

This protocol is used to recycle the correct tape from the recycled adhesive. The experiment for Gel recovery used the SanPrep Column DNA Gel Extraction Kit of sangon biotech

1. PREPARATION

To check whether absolute ethanol has been added to the Wash Solution. Check the Buffer B2 for precipitation Set the water bath pan to 50 °C.

• 1. Cut off the correct strip in the gel and put it into the EP tube
• 2. The cut gel was weighed, Add Buffer B2 to 300-600 µL per 100 mg agarose (100 mg).Then the centrifuge tube was placed in a 50°C water bath for 5-10 min and mixed well until the glue block is completely dissolved.
• 3. (Selected) For < 500 bp fragments, add 1/3 Buffer B2 volume of isopropanol.
• 4. The sol solution was moved into the adsorption column. 8000 g, Room temperature, 00:00:30 Remove the liquid from the collection tube.
• 5. Add in to 500 μL Wash Solution, 9000 g, Room temperature, 00:00:30 Remove the liquid from the collection tube.
• 6. Repeat step 5
• 7. The empty adsorption column was centrifuged at 9000 g for 1 minute. 9000 g, Room temperature.
• 8. The adsorption column was placed into a clean 1.5 mL centrifuge tube in suction 15-40 μL Elution Buffer was added to the center of the membrane and stood at room temperature After 1 min, they were centrifuged for 1 min. Save the DNA solution in the tube. 1 minute, 9000 g, Room temperature.
• 9. The concentration of DNA was detected at 340 nm using a spectrophotometer.
• 10. Store DNA at -20 °C.

Nucleic acid & protein electrophoresis

1. ABSTRACT

This protocol concludes two types of the electrophoresis used to detect target DNA or protein.

1. PREPARATION

Prepare 50X TAE, 1X Tris-Glycine electrophoresis solution, Coomassie blue staining solution, distaining solution before start.

• 1.Choose suitable electrophoresis method depends on the type of the sample.


• 2.Agarose gel electrophoresis: DNA was detected by agarose gel electrophoresis. (It is necessary to were latex gloves and mask when prepare the gel because most of the reagent are toxic. All the things used in the next steps should be washed after use and cannot be touched without wearing gloves. )

  2.1 Weigh appropriate agarose depends on the concentration of the gel (1% agarose gel for detection and 1% or 2% for gel extraction).

  2.2 Heat up by microwave until the solution is homogeneous.

  2.3 Cool at room temperature for 3~5 min. Let it cool down to 50-60 °C.

  2.4 Add 1 μL nucleic acid dye to the solution and shake the conical bottle to color.

  2.5 Put the solution into bed for polymerize, make sure "comb" is well placed and the solution is balanced.

  2.6 Wait about 20 min to let the gel completely concretes.

  2.7 Mix the sample with loading buffer and load the sample into the sample holes. (Our laboratory used 10X DNA loading buffer of Vazyme) Remember to load the DNA marker to the sample hole. (Our laboratory used DL 2000, DL 5000, and DL 15000 DNA Marker).

  2.8 Put the bed with gel into the electrophoresis chamber.

  2.9 Set the voltage of electrophoresis (80V~120V) and begin to run.

  2.10 Stop running when the front indicator reaches about 3/4 length of the gel.

  2.11 Electrophoresis was visualized with a Tanon Gel Imager.

3. SDS-PAGE: Protein was detected by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) (It is necessary to were latex gloves and mask when prepare the gel because most of the reagent are toxic. All the things used in the next steps should be washed after use and cannot be touched without wearing gloves. )

3.1 After a certain period of induction, the precipitate was obtained by centrifugation of 100 mL of the bacterial solution.

3.2 The bacteria were mixed with phosphate buffer and centrifuged. Centrifuge at 8000 g for 10 minutes at room temperature.

3.3 Add 20 mL of phosphate buffer and mix it well.

3.4 Put the EP tube on the ice and use ultrasonic cell disruptor to disrupt cells until the system becomes clear.

3.5 Centrifuge at 10000 g for 10 minute at 4 °C.

3.6 The supernatant was removed for cryopreservation. Remove 30 uL of supernatant and 7.5 μL of 4X Protein Sampling Buffer from the Solarbio and heat for 10 min in a metal bath at 95 °C. Store samples at -20 °C.

3.7 Wash glass plates and install the casting stand with the glass plates.

3.8 Inject the water into the pouring through to check leakage. The liquid level will drawdown in few minutes if the pouring trough has leakage in it. If the device leaks the liquid, it needs to be reassembled in time.

3.9 Pour out the water completely, you can take a paper towel to suck away the remaining water.

3.10 One-Step PAGE Gel Fast Preparation Kit of Vazyme (12%) to prepare protein gel. Prepare separating gel: Add 2.7 mL of each and 60 μl of APS of Resolver A and Resolver B to the beaker, mix and add to the device.Prepare the spacer gel: Add 0.75 mL and 15 μL APS of Stacker A and Stacker B to the beaker, mix and inject into the device. Inject the separation gel into the pouring through quickly to full the trough.

3.11 Insert the comb into pouring trough.

3.12 After the glue solidifies (about 15 min), the combed teeth can be used for electrophoresis.

3.13 Install the electric pool, install the gel with the glass plate into the electric pool, and pour in the Tris-Glycine electrophoresis buffer.

3.14 Load the sample (7-8 μL) and protein marker (2-3 μL) into the sample holes.

3.15 Run the gel at the voltage of 130V for about 90 min,Electrophoresis can be stopped when the bromophenol blue indicator reaches the bottom edge.

3.16 Stop running and peel the gel into the plastic box and clean it with distilled water (ddH2O).

3.17 Staining was performed using Coomassie Brilliant Blue Fast Staining solution for solarbio:Take 100 mL solution B, add 2 mL solution A, and mix well as the working solution.

3.18 Remove the SDS-PAGE gel (8cm 10 cm, for example) and put it into the container, add 50 mL of double steam or deionized water, heat to boiling and stop. (Optional: continue to shake on the decolorization shaker for 5 minutes), and discard the aqueous solution.

3.19 25 mL of rapid staining working solution was added, heated to boiling and kept boiling for 30-60 seconds, and continued after stopping heating Shake the shaker for 5-10 minutes and discard the staining solution (at this time the protein band should be visible).

3.20 Add about 50 mL of water, heat to boiling and keep in a boiling state for 30-60 seconds. After stopping heating, continue to shake on the decolorization shaker 5-10 minutes, change the water to complete the decolorization, observe the results.

Plasmid DNA extraction

1. ABSTRACT

This protocol is used to extract plasmid DNA from E. coli.

1. PREPARATION

Buffer P1; Buffer P2; Buffer P3; Buffer DW1; Wash Solution; Elution Buffer; RNase A;
(Visual Lyse.RNase A is stored at 2-8 °C and stored at -20 °C for long periods of time. The kit is shipped at room temperature. After adding RNase A, Buffer P1 should be stored at 2-8 °C for half a year. Other components can be stored at room temperature for one year, and for long-term storage, please store at 2-8 °C.)

• 1.Preparations. Check if RNase A has been added to Buffer P1. Check that absolute ethanol has been added to the Wash Solution. Check Buffer P2 and P3 for precipitation.
• 2.Collect the bacteria by centrifuging 1.5-5 mL of the bacteria cultured overnight, centrifuge 8,000 g for 2 minutes, and discard the medium.
• 3.Add 250 μL of Buffer P1 to the pellet and suspend the bacteria thoroughly.
• 4.Add 250 μL of Buffer P2 and immediately mix by gently inverting the tube 5-10 times. Let stand at room temperature for 2-4 minutes.
• 5.Add 350 μL of Buffer P3 and immediately gently invert the centrifuge tube 5-10 times to mix.
• 6.Centrifuge at 12000 g for 5-10 min. Transfer the supernatant into the adsorption column, centrifuge at 8000 g for 30 sec, and drain the liquid from the collection tube.
• 7.Add 500 μL of Buffer DW1, centrifuge 9,000 g for 30 sec and drain the liquid from the collection tube.
• 8.Add 500 μL of Wash Solution, centrifuge 9000 g for 30 sec, and decant the liquid from the collection tube.
• 9.Repeat step 8 once
• 10.Centrifuge the empty adsorption column at 9000 g for 1 min.
• 11.Place the adsorption column in a clean 1.5 mL centrifuge tube, add 50-100 μL of Elution Buffer to the center of the adsorption membrane, let stand at room temperature for 1 min, and centrifuge for 1 min. Save the DNA solution in the tube
• 12.Test the concentration and purity of DNA using NanoDrop.
• 13.Store DNA at -20 °C.

Polymerase chain reaction (PCR)

1. ABSTRACT

This protocol is used to amplify target DNA fragment for plasmid construction or other use. Set up a small box with ice, put DNA and Primestar (Takara) into it before going into the thermocycle.

1. PREPARATION

a. Primestar b. Template c. Forward Primer (10 μM) d. Reverse Primer (10 μM) e. ddH2O.PCR program as follows:

Program A was used for constructing plasmids.

PCR system(25 μL)
primestar	12.5 μL
forward primer(F)	1 μL
reverse primer (R)	1 μL
wild type	1 μL
DD H2O	up to 25 μL

A
95.0 °C, 05min00s
95.0 °C, 00min30s
55.0 °C, 00min30s
72.0 °C, 01min00s
Goto 2, x30
72.0 °C, 10min00s
4.0 °C, Forever

Program B was used for constructing plasmids.

PCR system(25 μL)
primestar	12.5 μL
primestar	12.5 μL
Fa+Fa'——>Fa''/Ra+Ra'——>Ra''	1 μL
wild type	1 μL
DD H2O	up to 25 μL

Fa+Fa'——>Fa'' Mix Fa and Fa' as primer Fa'' for PCR; Ra+Ra'——>Ra'' Mix Ra and Ra' as primer Ra'' for PCR

B Step 1	B Step 2
98.0 °C, 05min00s
98.0 °C, 00min15s
60.0 °C, 00min15s	55.0 °C, 00min15s
72.0 °C, 04min00s
Goto 2, X 3	Goto 2, X 15
72.0 °C, 10min00s
4.0 °C, Forever