Magarose^® ultra-low electroosmotic agarose

Agarose with ultra-low EEO

1. Product description

Magarose^® agarose series products are equipped with innovative second-generation desulfurization technology, breaking through the shackles of natural seaweed raw materials and realizing the free regulation of core indicators such as gel strength and electroostosis through molecular-level fine control processes.

Magarose^® ultra-low electroosmotonous agarose has extremely low electroosmosis (EEO=0.03), which makes the electrophoretic mobility of DNA significantly higher than that of traditional agarose gels, reducing the impact of band diffusion and improving the resolution of electrophoresis, which is very suitable for conventional nucleic acids, as well as convective immunoelectrophoresis and cross-immunoelectrophoresis.

2. Basic parameters

3. Product specifications

4. Transportation and preservation methods

5. How to use

Pulsed field gel electrophoresis experimental steps:

1. Preparation of DNA samples

To avoid breakage of macromolecular DNA during extraction, cells need to be lysed in situ in an agarose gel block, and Staphylococcus aureus was used in this experiment aureus) as an example.

1) Centrifuge 100 mL of logarithmic growth phase Staphylococcus aureus cells at 4 °C at 5000 g for 5 min.

2) Rinse the pellet with 20 mL of TEN buffer and centrifuge under the same conditions for 5 min. Cells are then suspended with 10 mL of EC buffer.

3) Take 1.5 mL of cell samples and the same volume of EC buffer containing 2% low melting point agarose (Cat. No. AG0065LM), mix and isolate the solution into a closed module to solidify at 4°C, heat to agarose to melt and cool to 50°C before mixing.

4) For each strain, 15~20 gel blocks are required and placed in 10 mL EC buffer containing 30~45 μg/mL RNase (50 mL of 10 mg/mL RNase) and shaken overnight at 37 °C.

5) Remove the lysis buffer and replace it with 10 mL of ESP buffer at 50 °C for 48 h.

6) Inactivate protease K in ESP by placing the gel block in 10 mL of TE buffer containing 174 μg/mL of benzethylsulfonylfluoride (PMSF) for 4 h (one 2 h change of fluid) at room temperature.

7) Wash the agar block with TE for 6 h (change once at 2 h) and store it in TE at 4 °C.

2. Restrict enzymatic digestion

Improving the resolution of PFGE depends on the reproducibility of the 100~1000 kb fragment electrophoresis results, which is closely related to digestion and can be digested using appropriate endonucleases in the agarose gel block.

1) 25 μL of 10× reaction buffer, 30 U of restriction enzyme, and double distilled water to 250 μL to mix.

2) Take a gel block and incubate it overnight at a suitable temperature (according to endonuclease requirements), then wash it with TE buffer and store it.

3. Analyze the sample DNA using PFGE

1) Prepare a 0×.8% Radical^TM HGT hypoelectrototonic agarose (Cat.No. AG2000LE) Gel, the thickness of the gel should be as consistent as possible with the sample gel block.

2) After gelling, carefully remove the sample comb. Cut the gel block with a knife to the same size as the well, and carefully insert the sample into the well, avoiding bubbles. (If the sample is in solution, mix it with 50 °C, 2% low melting point agarose (Cat. No. AG0065LM) phase mixed, and quickly injected into the dosing well).

3) Put the glue into the electrophoresis tank, add buffer, just cover the surface of the glue, and cool the buffer at 4 °C in advance.

4) Connect the electrophoresis tank to a programmed switchgear connected to a regulated power supply. Turn on the power supply and adjust the peristaltic pump to the appropriate flow rate (5~10 mL/min) or turn on the variable speed pump to 40.

5) Start the polarity conversion program through your computer. Restriction fragments greater than 50 kb were separated under forward and reverse electrical pulses of 1.2 s and 0.4 s, generally for 3.5 h or more. Restriction fragments of less than 50 kb were separated under electrical pulses (ratio of 2:1) in the forward direction of 0.4 s and reverse direction of 0.2 s for a time of 3~5 h.

6) Staining in 0.5 μg/ml ethidium bromide ingots and photographing.

4. Isolate and purify large DNA fragments

1) After gel staining and analysis, cut a groove of about 0.25^cm2 in front of the target band (near the positive electrode), and inject 0.8% low melting point agarose (Cat. No. AG0065LM) to solidify it.

2) Re-turn the polarity transfer switch on, detect the migration of DNA with UV transmitters, turn off the switch when the target band enters the gel tank, cut off the target band, and move it to a 1.5 mL EP tube.

3) Add 2 μL of tRNA, 30 μL of 5 mol/L NaCl, and 370 μL of distilled water at 65~70 °C and melt the gel and keep warm for 10 minutes.

4) Phenol/chloroform 500 μL extraction twice.

5) Preserve the aqueous phase with 2.5 times the volume of 95% ethanol and 1/10 volume of NaAc (3mol/L, pH 5.2) at -70 °C for 10 min. Wash twice with 70% ethanol and dissolve the pellet in TE buffer.

6. Precautions

1. This product is only for scientific research use and should not be used in clinical diagnosis and treatment.

2. Always wear goggles to protect yourself and others from burns when microwaving to dissolve agarose.

3. Be as careful as possible not to damage the agar gel when changing the buffer.

4. PMSF is a strong protease covalent inhibitor, that is, toxic and volatile, and should be operated in a fume hood.

5. Some high-voltage power supplies are not suitable for pulsed electric field gel electrophoresis, because they are designed with a protection circuit that can detect a sudden drop in load and trigger an automatic shutdown of the external power supply.

6. Due to the high voltage, heat will be generated, and some cooling equipment is needed to ensure the temperature is 14 °C. In addition, the electrophoresis system can be kept at a constant temperature by placing it in an open ice box.

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