Transfection of hippocampal neuronal cells with calcium phosphate co-precipitated plasmid DNA
Transfection of hippocampal neuronal cells with calcium phosphate co-precipitated plasmid DNA
The use of DNA/phosphorothioate (CAPO 4) co-precipitation to transfect cells is a widely described method. Although this co-precipitation method was proposed more than 30 years ago (Graham and van der Eb 1973), it is still widely used in laboratories today, mainly because of its obvious advantages over other transfection methods. Relative to commercially available transfection kits, this method requires inexpensive reagents, can be used for transfection experiments with a variety of established cell lines (both walled and unwalled) and primary cells, and can be used to construct stably transfected cell lines. Author: T. Friedman et al, Translated by W. Qin et al. This experiment is from "Gene Transfer".
Operation method
Calcium phosphate-mediated cell transfection Move Calcium phosphate-mediated cell transfection Materials reagents 50m m o l / L B E S I. 5 m m o l / L N a 2H P O 4 280 m m o l / L N a C l Adjust the pH of the buffers , choosing buffers with at least three pHs; use a freshly calibrated and clean p H meter. Use the 2XBBS working solution as a p H reference to prepare the next set of 2XBBS buffers. Sterilize the buffers by filtration, seal in a 50 ml glass vial and store at 4°C. The buffers can be stored for up to I2 months. The buffer can be stored for up to I2 months. Cell lines to be transfected (primary hippocampal neuronal cells) H BSS Wash (p H 7. 3) 135 m m o l / L NACL 20 m m o l / L H E P E S 4 m m o l / L K C l l m m o l / L N a 2H P O 4 2m m o l / L C a C l2 l m m o l / L M g C l2 10 m m l/ L glucose solution Adjust p H to 7 . 3, sterilized by filtration and stored at 4C for one year. 2.5m o l / L CaClz From our experience, repeated freezing of 2 XBBS buffer and CaClz solution can affect the reproducibility of transfection results. Therefore, we recommend that all solutions and buffers be stored at 4°C N M E M -B 2 7 Transfection medium (p H 7. 4) Add lmmol/L sodium propionate (Sigma-Aldrich), 15mmol/L HEPES (Invitrogen), 2mmol/L L-glutamic acid (PromoCell), 1XB27 supplement (Invitrogen) and 33mmol/L I> to MEM (Invitrogen). Plasmid DNA To prepare plasmids of high purity, use an endomycin-free Maxi-prep kit (e.g. QIA G EN Endomycin-Free Maxi Kit) or a CsCl gradient. The purity of the DNA is evaluated by measuring the OD value, O IW O D 28. An OD value of approximately 1.8 is optimal. Dissolve the plasmid in water or TE buffer. Centrifuge the plasmids at 4T before use. Two plasmids can be used for cotransfection. Typically, when the ratio of two given plasmids is 1 : 1, the efficiency of cotransfection is up to 95 % (GoetzeetaL 2004). Transfection with three or more plasmids at a time is more difficult and must be carefully controlled at the single-cell level using appropriate markers. Instrumentation Freshly calibrated and clean PH meter. Methods 1- Prepare the cells to be transfected by transferring them to a glass culture dish containing 2m l of transfection solution. 2- To prepare the co-precipitation, transfer the following in order in a 1.5m l centrifuge tube: 6ul CaCl2 x ul H 2O (mix well) 3ug plasmid D N A (add D N A slowly while mixing well with a lance tip) The total volume is 60ul, and the insufficient part is made up with water. 3- Add 60ul of 2 X B B S buffer dropwise to the CaCl2/D N A solution. Gently tap the centrifuge tube 5 times after each 3-drop addition to mix the components well. Do not vortex! 4 . Quickly add 1200ul of transfection mixture to the transfection medium. Incubate with the cells at 36. 5°C, 5 % C O 2 in a cell culture incubator. In order to increase the amount of coprecipitate reaching the cell surface, when the precipitate can be observed with the naked eye, centrifuge the culture dish containing the cells at 1000 r/min for 2 to 6 min at room temperature, and observe the cells every 10 to 15 min during subsequent incubation, as the cell death rate increases rapidly with time. Cell death is typically characterized by cell lysis and loss of nuclear integrity. When centrifugation is used, the co-cultivation time of centrifuged cells with sediment can be significantly shortened and the transfection efficiency increased by 50 % compared to cells without centrifugation (B. Goetze etal., unpublished). After 5.45mirx or until 6h , the neuronal cells are uniformly covered by fine co-precipitates, which can be clearly observed with an I O X objective (Goetze et al. 2004). 6- Aspirate the transfection culture solution and replace it with prewarmed H B S S wash solution. Observe the cells under the microscope after 5m i n , by which time the precipitate should have completely dissolved. Do not wash the cells for more than 15m m . 7 - Aspirate the H B S S buffer to remove the D N A /C a P 0 4 precipitate and incubate the cells in normal medium. 8- Detect protein expression by suitable methods (depending on the experimental design, methods such as detection of reported gene expression, staining and microscopy, immunostaining, and immunoblotting can be used) . For more product details, please visit Aladdin Scientific website.
2XBES Buffered Biological Saline (BBS) (pH 6.85 to 7.20)
glucose (Sigma-Aldrich). Adjust p H to 7.4, sterilize by filtration and store in 50 ml sealed glass vials for 2 months.