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1. Reagent preparation

Sterile water ddw: high temperature sterilization; dispensing;

2XHBS: 280 mM NaCl

10mM KCl

1.5 mM Na2HPO4

12 mM glucose

50 mM HEPES

Adjust the pH , every 0.05pH from 7.00 to 7.45 using 10N NaOH, then add ddw. to the final volume. Filter sterilization, dispensing;

2M CaCl2: Filter sterilized and dispensed.

2. Experimental process:

1. Laying cells: Choosing well-conditioned 293T cells, 2-3 x 105 cells/35 mm dish.

2. After 20-24h, transfection is performed when the cells grow to about 50-70% of the bottom of the bottle. Take the 35mm dish as an example below, using the following transfection system:

Ddw: 105ul

Plasmid: 2 ug (eg 0.5ug/ul, ie 4ul)

2M CaCl2: 16.5ul

2XHBS: 125ul

The above four reagents were sequentially added to the eppendorf tube in the above order.

First mix the first three, and finally add 2XHBS.

One method is to add 2XHBS dropwise, blow it to a slightly milky white, immediately drip into the Petri dish, gently shake it, place it in the incubator, and change the solution after 6-8h.

Another method is to blow about 2 times immediately after adding 2XHBS (however, this depends on the strength of each person and the frequency of blowing. It is recommended to do a gradient experiment, and blow different times to see which transfection efficiency is high. Press this number of times to blow), then the steps are the same as above, immediately drip into the Petri dish, gently shake and place in the incubator, and change the solution after 6-8h.

Surfactants

We adopt a comprehensive classification method, which is divided by the ionicity of surfactants as follows which can all be provided by Insen Biotech: 1. Anionic surfactant: stearic acid, sodium dodecylbenzene sulfonate. 2. Cationic surfactant: quaternary ammonium compound. 3. Zwitterionic surfactants: lecithin, amino acid type, betaine type. 4. Non-ionic surfactants: fatty acid glycerides, fatty acid sorbitan (Span), polysorbate (Tween).

Surfactant Agent ,Carbomer 940 Powder,Surfactant Cosmetic ,Nonionic Surfactant

Hunan Insen Biotech Co., Ltd , https://www.insenhealth.com