Preparation method of transfection reagent for easy DNA combination
A technology of transfection reagent and polyethylenimine, which is applied in the direction of recombinant DNA technology and the use of vectors to introduce foreign genetic material, etc., can solve the problems of high molecular weight, high cytotoxicity, high cytotoxicity, etc., and achieve good biocompatibility, The effect of the fast and cheap operation of the method
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Embodiment 1
[0027] (1) Add 3g of branched PEI into 15mL of three-distilled water, heat and stir to make it dissolve rapidly, and obtain solution A;
[0028] (2) carbon dioxide (CO 2 ) into solution A, at room temperature, continued bubbling for 5h, and stirred to make the reaction fully, to obtain solution B. Put 2 mL of solution B into an EP tube, freeze-dry, and grind to obtain a modified yellow solid powder. Take 80mg of the yellow powder and branched PEI obtained according to the above steps and dissolve them in 1.5mL of D 2 O, do carbon nuclear magnetic resonance spectrum, the obtained results are as follows figure 1 shown.
Embodiment 2
[0030] (1) Gel electrophoresis experiments are used to evaluate the ability of the carrier to bind to the gene. Take 1 μg of pDNA and the appropriate yellow powder and hyperbranched PEI of Example 1 to prepare different N / P ratios (N / P=0, 0.5, 1, 1.5, 2, 3, 4, 5) PBS solution containing the complex and adjust the final volume to 3 μL, and add 1ul 6X loading buffer containing phenol blue (50% glycerol + 0.25% phenol blue).
[0031] (2) Prepare 0.8% agarose Tris-boric acid-EDTA (TBE) buffer solution pH=8.3, after the agarose gel is cooled and solidified, add the above-mentioned prepared mixed solution into the sample well. The electrophoresis was continued for 30 minutes at a voltage of 120 V, and the gel was taken out and stained with TBE solution with an EB concentration of 0.5 μg / mL.
[0032] (3) Gel Doc XR+ imaging system (Biorad Laboratories, Hercules, CA) was used for investigation. The result obtained is as figure 2 shown
Embodiment 3
[0034] (1) The yellow powder of Example 1 and hyperbranched PEI were configured into 30 mL of 150 mM NaCl aqueous solution with a polymer concentration of 0.2 mg / mL, and the pH value of the polymer solution was adjusted to around 2 with 0.1 M HCl solution. Under rapid stirring, the polymer solution was titrated with 0.1 M NaOH solution at room temperature, and the pH value of the solution was tested with a Sartorius PB-10 pH meter.
[0035] (2) Use Origin 8.0 to draw the measured pH value as a change curve with the volume increase of 0.1M NaOH, and the obtained results are as follows image 3 shown
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