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Description. Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been. Molecular Cloning, A Laboratory Manual, 4th Edition, prelinparbalym.cf Cold Spring Harbor Protocols, prelinparbalym.cf This is a free sample of.
Print Share. Add to cart. Specifications Documents. Specifications Specifications For Use With Application Manual explains how to achieve success in cloning and provides a wealth of information about why techniques work, how they were first developed and how they have evolved Category Biology Includes Laboratory protocols.
Sambrook, D. Provide Content Correction. Incubate the mixture as described above. Both types of reaction mixtures can be doubled or quadrupled in volume if a larger number of cells are to be transfected. When the volumes are quadrupled, a larger tube e. Normally, 0. Published procedures differ widely in the manner and rate of mixing of ingredients. Some advise against anything but the gentlest agitation and suggest that air, bubbled from an electric pipetting device, should be used to mix the solution. Others advocate continuous slow mixing during addition of the DNA solution, followed by gentle vortexing.
The object is to avoid the rapid formation of coarse precipitates that results in a decreased efficiency of transformation. In practice, several factors other than the speed of mixing affect the size of the precipitate, including the concentration and size of the DNA high-molecular- weight DNA can be sheared by passage through a fine syringe needle and the exact pH of the buffer some workers make up several batches of HBS buffer over the pH range 6.
If it is crucial to achieve the highest transfection efficiencies, time should be spent to optimize these factors for your particular system. Once an efficacious batch of reagents has been prepared and stored as indicated above, reproducible results can be obtained over long periods of time.
Transfer the calcium phosphate-DNA suspension into the medium above the cell monolayer. Use 0. Rock the dish gently to mix the medium, which will become yellow-orange and turbid. An alternative method is to remove the medium and add the precipitate directly to the cells.
Incubate the cells for 15 minutes at room temperature, and then add medium to the dish. The transfected cells can then be treated in one of the following ways: a. If no additional treatments with reagents such as chloroquine, glycerol, or sodium butyrate; see below are to be employed, incubate the cells for hours; then remove the medium and precipitate by aspiration.
Wash the monolayer once with phosphate-buffered saline PBS; see Appendix B , and add 5 ml of prewarmed complete growth medium. Return the cells to the incubator for hours before In many instances, uptake of DNA is increased by concurrent treat- ment of the cells with chloroquine, which may act by inhibiting the degradation of the DNA by lysosomal hydrolases Luthman and Mag- nusson The concentration of chloroquine to be added to the growth medium and the time of treatment are limited by the sensitivity of the cells to the toxic effect of the drug.
The optimal concentration of chloroquine for the particular cell type that is used should therefore be determined in preliminary experiments. However, most cell types respond well to treatment with chloroquine at a final concentration of p,M for 3—5 hours. A stock solution mw of chloroquine diphosphate is diluted directly into the medium either before or after the addition of the calcium phosphate— DNA coprecipitate to the cells see alternative procedures in step 4. During treatment with chloroquine, it is normal for cells to develop a vesicularized appearance.
After the 3— 5-hour treatment with DNA and chloroquine, remove the medium and the precipitate, wash with PBS, and add 5 ml of pre- warmed complete growth medium. Return the cells to the incubator for 24—60 hours before assaying for transient expression of the transfected DNA or replating the cells in appropriate selective medium for the isolation of stable transformants.
Brief treatment of transfected cells with glycerol has also been shown to increase the efficiency of transformation or transient expression of the introduced DNA. This procedure may be used following treatment with chloroquine. Because cells vary widely in their sensitivity to the toxic effects of glycerol, each cell type must be tested in advance to determine the optimum time 30 seconds to 3 minutes of treatment. Remove the growth medium by aspiration and wash the monolayer once with PBS.
Add 1. Remove the glycerol by aspiration, and wash the monolayers once with PBS. Add 5 ml of complete growth medium, and incubate the cells for 24—60 hours before assaying for transient expression of the trans- fected DNA or replating the cells in the appropriate selective medium for the isolation of stable transformants. Cells are exposed to sodium butyrate following glycerol shock by adding the agent directly Expression of Cloned Genes in Cultured Mammalian Cells Different concentrations of sodium butyrate prepared as a him stock solution by neutralizing butyric acid in a chemical hood with NaOH are used, depending on the cell type, for example: CV-1 10 mM NIH-3T3 7 ntM HeLa S3 5 mM CHO 2 mM Incubate the cells for hours before assaying for transient expres- sion or replating the cells in the appropriate selective medium for the isolation of stable transformants.
Newly synthesized protein may be analyzed by radioimmunoassay, by western blotting, by immunoprecipitation following in vivo metabolic labeling, or by assays of enzymatic activity in cell extracts.
hukusyuu.com/profile/2020-01-04/handy-verloren-orten-lassen-base.php For assays that involve replicate samples or treatment of transfected cells under multiple conditions or over a time course, it is desirable to avoid dish-to-dish variation in transfection efficiency. In these cases, it is best to transfect large monolayers of cells mm dishes and then to trypsinize the cells after 24 hours of incubation and distribute them among several smaller dishes.
Stable transformation: Following hours of incubation in non- selective medium to allow expression of the transferred genets to occur the cells are trypsinized and replated in the appropriate selective medium. This medium should be changed every days for weeks to remove the debris of dead cells and to allow colonies of resistant cells to grow. Individual colonies may be cloned and propagated for assay for methods, see Jakoby and Pastan Transfection of Adherent Cells in Suspension 1.
Prepare the solutions and form the calcium phosphate— DNA coprecipitate as described on pages While the precipitate is forming, harvest exponentially growing adherent cells by trypsinization. Discard the supernatant. Resuspend each aliquot of 10 6 cells in 0. To each aliquot, add 4. Incubate the cells for up to 24 hours depending on the choice of subsequent treatments; see step 5, pages Note This technique can easily be modified to accommodate greater numbers of cells.
After 15 minutes, the mixture was diluted with 40 ml of complete medium supplemented with 0. Discard the supernatant, and resuspend the cell pellet in 20 volumes of ice-cold phosphate-buffered saline PBS; see Appendix B. Divide the suspension into aliquots that contain 1 x 10 7 cells. Recover the washed cells by centrifugation, and again discard the supernatant. Add 10 ml of serum-containing medium with or without chloroquine; see page Recover the cells by centrifugation at g for 5 minutes at room tempera- ture, and wash them once with PBS.
Resuspend the cells in 10 ml of prewarmed complete growth medium.
Return the cells to the incubator for 48 hours before assaying for transient expression of transfected genes step 6a, page Note If the cells have been proved to survive treatment with glycerol, a glycerol shock detailed below may be used hours after exposure of the cells to the calcium phosphate-DNA coprecipitate begins in order to improve the frequency of transfection: 1. Collect the cells by centrifugation as described in step 5, and wash them once with PBS.
Dilute the suspension with 10 ml of PBS, and recover the cells by centrifugation as described in step 5. Wash the cells in PBS. Resuspend the cells in 10 ml of serum-containing medium, and plate them in a mm tissue culture dish. The calcium phosphate-DNA coprecipitate is allowed to form in the tissue culture medium during pro- longed incubation hours under controlled conditions of pH 6.
It should be noted that linear DNAs yield very low transformation frequencies using this protocol, perhaps because the slow formation of the calcium phosphate-DNA coprecipitate delays protection of the DNA from nucleases. The best results have been obtained using supercoiled plasmid DNAs purified by two rounds of equilibrium centrifugation in CsCl-ethidium bromide density gradients. Adjust the pH of the solution to 6. Twenty-four hours before transfection, harvest exponentially growing cells by trypsinization and replate aliquots of 5 x 10 5 cells onto mm tissue culture dishes.
Mix jug of superhelical plasmid DNA with 0. Do not expect a visible precipitate to form during this time.