简易纯化方法

Preparation of Viral DNA

A variety of methods are available for the preparation of viral DNA. These methods differ in the degree of purity of viral DNA that is obtained and generally increase in complexity with increasing purity. Therefore, for each individual application, it is necessary to determine the degree of purity required and, thus, the optimal method to be used. The methods available are described here in order of increasing purity of the DNA obtained.

Preparation of Total DNA from Infected Cells

For many applications, such as checking the structure of a recombinant virus, a crude DNA preparation containing cellular as well as viral DNA is quite sufficient. Thus, if total DNA is prepared from infected cells showing a cytopathic effect, and digested with appropriate restriction enzymes, viral DNA bands are readily seen following gel electrophoresis within the smear of cellular DNA The structure and size of particular regions of the viral DNA genome can readily be determined by Southern blotting of this DNA preparation and hybridizing with appropriate probes. As a negative control, DNA can also be prepared from uninfected cells and included in the blot hybridization.

The method used in our laboratory to isolate total DNA from infected cells is given below. A similar method has been used by De Luca et al.

1、Infect 107 cells at 0.1 PFU/cell and leave at 37°C until a cytopathic effect is visible.

2、Remove medium and wash the cells in PBS.

3、Scrape the cells into the PBS and pellet by low-speed centrifugation (3000 g for 10 min).

4、Incubate the cells at 37°C overnight in PEST buffer.

5、Extract the solution once with phenol and once with phenol/chloroform/isoamylalcohol (25:24:1).

6、Precipitate by adding of 2 vol of ice-cold ethanol, and leave overnight at -20°C.

7、The DNA is harvested by centrifugation (10000g for 20 min), washed once in 70% ethanol, dried under vacuum, and gently resuspended in an appropriate vol of TE. The DNA is now ready for digestion with restriction enzymes or for other applications.

也可以直接买提病毒DNA的试剂盒，效果很好，又方便，价格大概50T/2000元。

病毒滴定测定

Once the virus stock has been obtained, its infectivity must be assayed before it can be used in experiments. If it is thought that viral titer will vary considerably between cell types, it is necessary to titrate the virus in the cell type in which it will eventually be used. For routine applications however, we titrate the virus in a monolayer assay on Vero cells, which normally results in the appearance of large, easily counted plaques. Whatever cell line is employed, it is necessary that the conditions that are used allow the cells to maintain a confluent monolayer for the time taken for plaques to appear, i.e., a mimimum of 2–3 d, and up to a week for some temperature-sensitive mutants. This can be achieved by reducing the level of serum in the medium and is also critically dependent on the brand of plasticware used, which affects the ability of the cells to remain adherent for long periods. The conditions described below work well for Vero cells and for all viral strains tested.

1、Seed 106 Vero cells/well into 35-mm wells (use Flow, Linbro 6-well dishes) 24 h prior to use. This should give a confluent monolayer when required.

2、 Remove the medium and add 0.1 mL of each virus dilution in serum-free medium to individual wells and leave for 1 h to adsorb.

3、Add 5 mL of medium containing 2% fetal calf serum and 0.5% carboxymethyl cellulose. Avoid disturbance and incubate for several days (normally 2–3 d) until discernable plaques appear.

4、 When plaques are visible, remove the medium and rinse the wells gently to remove all the carboxymethyl cellulose.

5、Fix the cells briefly in methanol/glacial acetic acid (3:1).

6、Stain cells briefly with a solution of crystal violet (0.7%) in 50:50

7、Plaques can be counted by eye or with the aid of a low-power objective. Typical plaques are illustrated in Fig

8、Viral liter is calculated on the basis of the number of plaques formed in the various dilutions.