藥學(xué)專業(yè)畢業(yè)論文外文翻譯--聚丙烯酰胺凝膠上dna銀染法（英文）

1、Silver staining DNA in polyacrylamide gelsBrant J Bassam1 doi:10.1038/nprot.2007.330This protocol describes a simple silver staining method used to visualize DNA fragments and other organic molecules with unsurpassed de

2、tail following traditional polyacrylamide gel electrophoresis (PAGE). Sensitivity rivals radioisotopic methods and DNA in the picogram range can be reliably detected. The described protocol is fast (B1 h) and is implemen

3、ted using readily available chemicals and materials. To achieve the sensitivity and visual clarity expected, quality reagents and clean handling are important. The updated protocol described here is based on the widely u

4、sed method of Bassam et al. (1991), but provides improved image contrast and less risk of staining artefacts.INTRODUCTION The separation of complex DNA samples and other biological molecules with high resolution by polya

5、crylamide gel electrophor- esis (PAGE) has broad application. However, to realize the potential of PAGE, a visualization method offering superior clarity and sensitivity is also required. The silver staining method descr

6、ibed here has proven very effective in this regard. As a method, silver staining was originally developed to detect proteins separated by PAGE1–3. It was further optimized and applied to visualize other biological molecu

7、les, for example, nucleic acids4,5, lipopolysaccharides6, glycoproteins and polysaccharides7. These earlier protocols were, however, comparatively tedious and offered limited sensitivity. The development of DNA amplifica

8、tion fingerprinting (DAF) by Caetano-Anolle ´s et al.8 required a superior protocol to adequately resolve and visualize complex DNA profiles. These requirements led directly to the codevelopment of a successful comb

9、ination of polyester-backed PAGE gels and DNA silver staining. The silver stain protocol developed for DAF was described separately by Bassam et al.,9 and has since gained wide acceptance including commercialization (e.g

10、., in the GenePrint STR systems and SILVER SEQUENCE products from Promega Corporation, USA). Silver staining of DNA (and other biological samples) has several advantages: ? Image development and visualization is done und

11、er normal ambient light. Thus, the procedure can be performed entirely at the laboratory bench without the need for darkroom or UV illumination facilities. ? The image is resolved with the best possible sensitivity and d

12、etail, because silver is deposited directly on the molecules within thetransparent gel matrix. Thus visualization is from the primary source and does not suffer any degradation or blurring that can accompany secondary im

13、aging devices which involve fluores- cence, autoradiography, focusing lenses, film development or digital image processing. ? Silver staining offers similar sensitivity to autoradiography, but avoids radioactive handling

14、, delays from development times and waste disposal issues. ? As a preferred option, gels can be dried onto a semi-rigid plastic backing film such as GelBond PAG film, creating a permanent record of the original material8

15、 (see ref. 10 for a revised protocol). Air-dried gels are resilient, preserving a concentrated and contrast-intensified image. They can also be stored indefi- nitely without distortion, obviating the need and added expen

16、se of photography and printing. In addition, the preserved gel is a ‘molecular archive’ , as stained DNA bands are ‘real’ DNA that can be extracted, amplified, cloned and DNA-sequenced.While the protocol described here f

17、ocuses on the visualization of DNA by PAGE, the method can be used without modification to visualize a wide range of biological molecules including RNA, polysaccharides, lipopolysaccharides, proteins and polypeptides in

18、polyacrylamide gels (data not shown). Although this is an advantage in terms of scope, it nevertheless means that the protocol must be applied with due care; stray human fingerprints on the gel surface stain with perfect

19、 detail as will almost any other biological impurity incorporated into or onto the gel matrix. It is thus important to use dust-free reagents of the best analytical grade, including the purest water available.MATERIALS R

20、EAGENTS .Acetic acid, glacial, AR Select (ACS) (CH3COOH; e.g., Mallinckrodt, cat. no. v193) m CRITICAL For best results, must be of high purity. .Silver nitrate crystal, AR (ACS) (AgNO3; e.g., Malinckrodt, cat. no. 2169)

21、 (see REAGENT SETUP) m CRITICAL For best results, must be of high purity. .Formaldehyde solution, AR (ACS) (HCHO; e.g., Malinckrodt, cat. no. 5016) (see REAGENT SETUP) m CRITICAL For best results, must be of high purity.

22、 .Sodium thiosulfate (Na2S2O3; e.g., Sigma, cat. no. S7026) (see REAGENT SETUP) m CRITICAL For best results, must be of high purity..Sodium carbonate powder, Z99.5%, ACS reagent (Na2CO3; e.g., Sigma-Aldrich, cat. no. 223

23、530) m CRITICAL For best results, must be of high purity. .Ethanol .Polyacrylamide gel .Fixer solution (see REAGENT SETUP) .Developer solution (see REAGENT SETUP) .Developer stop solution (see REAGENT SETUP) EQUIPMENT .P

24、lastic storage carboys with spigot (from any general laboratory supplier)p u o r Gg n i h s i l b u Pe r u t a N7 0 0 2 © natureprotocols / m o c . e r u t a n . w w w / / : p t t hNATURE PROTOCOLS | VOL.2 NO.11 | 2

25、007 | 2649PROTOCOL11| To wash the gel, pour sufficient deionized water into the staining tray to cover the gel to a depth of B5 mm.12| Rock the staining tray continuously on a platform rocker for 2 min. Longer times may

26、be needed if gels thicker than B1 mm are used. If the gel is washed for too long (over B20 min), then staining may be compromised, and fainter bands will result.13| At the end of the wash, carefully decant the wash solut

27、ion as described in Step 10.14| Repeat the wash steps two times more for a total of three washes in deionized water. m CRITICAL STEP Washing the gel is important. It removes acid and other trace substances that interfere

28、 with staining, and provides a clear, blemish-free background to the final stain.Formaldehyde pre-treatment 15| Add sufficient formaldehyde solution to cover the gel in the staining tray to a depth of B5 mm. Gently rock

29、the staining tray continuously on a platform rocker. For typical mini-gels of B1 mm thickness, a minimum of 5 min formaldehyde pre-treatment is required while B10 min provides optimum contrast. Longer times may be needed

30、 if thicker gels are used. This step may continue for up to B30 min. As an alternative, formaldehyde pre-treatment (Step 15) and silver impregnation (Step 17) can be combined as per the original Bassam et al.9 protocol.

31、See ANTICIPATED RESULTS to view an example gel processed using this alternative. m CRITICAL STEP Formaldehyde pre-treatment is important for stain sensitivity and maximum image contrast.16| Following the formaldehyde pre

32、-treatment, carefully decant the solution, taking care not to damage the gel or touch the gel surface. Alternatively, use a water-powered Venturi pump to vacuum the fixer solution away and down the sink.Silver impregnati

33、on 17| Add sufficient silver solution to cover the gel in the staining tray to a depth of B5 mm.18| Gently rock the staining tray continuously on a platform rocker. For typical mini-gels of B1 mm thickness, a 20 min impr

34、egnation time is usually optimal. m CRITICAL STEP The recommended silver concentration cannot be reduced without affecting sensitivity and contrast. A careful examination of silver impregnation times showed that optimal

35、staining was achieved after B20 min. However, as little as 10 min is sufficient for high-quality staining without significant loss of sensitivity. Impregnation times can be increased up to B60 min, but greater than B90 m

36、in can cause severe image loss.19| Following silver impregnation, carefully decant the solution, taking care not to damage the gel or touch the gel surface. ! CAUTION The silver solution is toxic and should be disposed o

37、f with care. Avoid spilling the solution, as it will permanently stain most surfaces. We precipitate the silver from used silver solution with NaCl in glass Winchester bottles, and accumulate it for recycling (it should

38、turn milky in the bottle as the silver precipitates).20| Briefly rinse residual silver solution from the surface of the gel by rinsing with B100 ml of deionized water for 5–10 s. Do not rinse the gel longer than B15 s, a

39、s this step removes silver from the gel.Image development 21| Check whether the developer is cold (it should be between 4 and 10 1C). Add sufficient developer solution to cover the gel in the staining tray to a depth of

40、B5 mm. Agitate the staining tray throughout image development so the developer solution is not stagnant. Image development begins as soon as the developer solution is added. The developer solution is kept cold to control

41、 the rate of image development, since development is usually too fast to control if done at temperatures above 10 1C. Image development typically takes about 3 min depending on gel thickness, the reagents used and the te

42、mperature of the reagents. Alternatively, you can add 600 ml of formaldehyde solution per 100 ml of final developer solution as per the original method9. This may improve image contrast, but usually also increases the ba

43、ckground and edge-staining artefacts. See ANTICIPATED RESULTS to view an example gel processed using this alternative. Room temperature development is possible for thin gels (less than B1 mm in thickness). See ANTICIPATE

44、D RESULTS to view an example gel processed using room temperature reagent alternatives. m CRITICAL STEP Decreasing Na2CO3 concentration below the recommended levels causes higher background staining and poor image contra

45、st. Poor staining can also result from the use of low quality or old (stale) reagents.Stopping the reaction 22| Decant the developer solution carefully, avoiding damage to the gel or touching the gel surface.23| Check wh

46、ether the developer stop solution is cold (it should be stored refrigerated at 4 1C). Add sufficient developer stop solution to cover the gel in the staining tray to a depth of B5 mm. As an alternative, developer stop so

47、lution kept at room temperature can be used for thin gels (o1 mm in thickness). However, this alternative requires some practice as thep u o r Gg n i h s i l b u Pe r u t a N7 0 0 2 © natureprotocols / m o c . e r u