M5GelredPlus核酸染料更清晰的EB替代品使用說明書

1、M5 Gelred Plus核酸染料（更清晰的EB替代品）使用說明書產(chǎn)品名稱 單位 貨號M5 Gelred Plus核酸染料(10000X) 500l MF079-plus-01M5 Gelred Plus 核酸染料(10000X) 5x500l MF079-plus-05【儲存條件】2-8C（避免太陽光直射）?！井a(chǎn)品組分】 MF079-plus-01 MF079-plus-05M5 Gelred Plus核酸染料(10000X) 500l 5x500l【產(chǎn)品簡介】M5 Gelred Plus核酸染料(10000X)是在基礎(chǔ)上優(yōu)化開發(fā)的一種具有凝膠染色特性，并被設(shè)計(jì)為替換高毒性染色劑溴化乙錠（

2、EB）的紅色熒光核酸染色劑，比具有更高的清晰度和靈敏性。 因?yàn)镚elred Plus與EB有著相同的光譜特性，可以在不改變?nèi)魏纬上裣到y(tǒng)的情況下用來替換EB。如果使用的是SYBR（如SYBR Green 1/SYBR Gold)染色劑，并使用紫外透射器(UV transilluminator）來觀察凝膠，那可以使用 Gelred Plus替換SYBR染色劑，不需要更換現(xiàn)有的SYBR慮光片。然而，在488 nm 激光或類似可見光下 Plus不能被充分地激發(fā)，如果需要，建議您使用GelGreen染色劑，其靈敏度與SYBR Green I一樣，但其穩(wěn)定性和可靠性遠(yuǎn)勝于后者。 Plus既可用于前染 (p

3、recast gel staining)，也可用于后染 (post gel staining)。通常后染比前染能夠獲得更靈敏的特性，并能排除染色劑在電泳過程中對核酸條帶分離造成任何影響的可能性。然而，前染較后染更為簡單、經(jīng)濟(jì)，因?yàn)榍叭静恍枰~外的著色過程，并且染料用量更少。另外，與GelGreen 、 EvaGreen 一樣，相對EB或SYBR， Plus誘導(dǎo)突變的能力極低。 M5 Gelred Plus核酸染料, 10,000X in water為濃縮的 Gelred Plus溶液。用于前染時，可稀釋10,000倍后使用；用于后染時，建議您稀釋3,300倍后使用，見具體操作步驟?！井a(chǎn)品特點(diǎn)】

4、1. 安全無毒： 獨(dú)特的油性大分子特點(diǎn)使其不能穿透細(xì)胞膜進(jìn)入細(xì)胞內(nèi)，艾姆斯氏試驗(yàn)結(jié)果也表明該染料的 誘變性遠(yuǎn)小于 EB。 2. 靈敏度高：適用于各種大小片段的電泳染色，對核酸遷移的影響較小。樣品熒光信號強(qiáng)，背景信號低。 3. 穩(wěn)定性高：適用于使用微波或其它加熱方法制備瓊脂糖凝膠；室溫下在酸或堿緩沖液中極其穩(wěn)定，耐光性強(qiáng)。而且不揮發(fā)！ 4. 操作簡單：在預(yù)制膠和電泳過程中不降解，可直接用可見光凝膠透射儀觀察。 5. 適用范圍廣：可選擇電泳前染色（膠染法）或電泳后染色（泡染法）；適用于瓊脂糖凝膠或聚丙烯酰胺凝膠電泳；可用于 dsDNA、ssDNA或 RNA 染色。 【操作步驟】一、膠染法（前染法）

5、（用法同EB）1按常規(guī)操作，制備瓊脂糖凝膠，加入濃縮的10000X Gelred Plus， 使其在凝膠中的終濃度為1X Gelred（比如，制備100ml凝膠，加入染料5l-10l，可根據(jù)實(shí)際情況調(diào)整用量），輕輕搖勻，倒膠。2. 因?yàn)榉浅ｌ`敏，電泳過程中 DNA marker 上樣量只需1-2ul，而不是EB電泳中的5ul，請嚴(yán)控DNAmarker上樣量。3. 按常規(guī)方法電泳，觀測結(jié)果。二、泡染法（后染法，具體方法見背面）1. 按照常規(guī)方法進(jìn)行電泳。2用dH2O將10000X Gelred Plus濃縮液稀釋約3300倍到0.1M的NaCl中，制成3X染色液。（比如，將15l 10000X

6、Gelred Plus濃縮液和5ml 1M NaCl加到45ml dH2O中）。3. 將凝膠小心放入合適的容器中，緩慢加入足量的3X染色液浸沒膠。室溫振蕩染色約10-30min，最佳染色時間根據(jù)凝膠厚度及瓊脂糖濃度不同而略有不同。對于3.5-10%丙烯酰胺膠，染色時間通常介于30min到1小時。然后觀測結(jié)果?！靖戒洠汉笕灸z標(biāo)準(zhǔn)操作流程】核酸電泳后染膠因?yàn)槲廴緟^(qū)域小，污染操作可控，越來越得到實(shí)驗(yàn)室的接受和采用。標(biāo)準(zhǔn)操作步驟（以配100ml 1%的瓊脂糖為例）：1、 稱1g瓊脂糖，量取100m l 1xTAE（或1x TBE）電泳緩沖液， 依次倒入一個三角瓶中。2、 在微波爐中化膠煮沸致瓊脂糖完全

7、融化。3、 取出靜置5分鐘，待膠液溫度降至50度，將膠液倒入制膠模上。4、 20分鐘后待膠完全成型，取出放入電泳槽。5、 將PCR產(chǎn)物或者其他DNA樣品和上樣緩沖液混合，逐一上樣。6、 電泳20-30分鐘，根據(jù)溴酚藍(lán)位置判斷電泳到合適時間，停止電泳。7、 將跑完電泳的膠放入含有染料的液體中，染膠10分鐘（如果膠厚適度延長時間）。8、 取出膠，放入掃膠儀中觀察結(jié)果。染膠液的配置：180ml dH2O中加入20ml 1M NaCl，再加入10000x Gelred濃縮液 60ul，也可以向聚合美購買即用型1x 染膠液（貨號：MF834-01，500ml）染膠液的使用：配置好的染膠液可以重復(fù)使用很多

8、次，直至染膠強(qiáng)度很低再重新配置。GelRed Nucleic Acid Gel StainDescriptionGelRed Nucleic Acid Gel Stain is a kind of new generation of fluorescent nucleic acid gel stain designed to replace the highly toxic ethidium bromide (EtBr). The Ames test confirmed that Gelgreen are nonmutagenic at concentrations well above th

9、eir working concentrations used for gel staining. Gelgreen Nucleic Acid Gel Stain is highly sensitive than EtBr either as precast gel stains or post gel stains.GelRed and EB have virtually the same spectra, so you can directly replace EB with GelRed without changing your existing imaging system. Gel

10、Red cannot be sufficiently excited with a 488nm argon laser or similar visible light. GelRedcan also be used to stain dsDNA, ssDNA or RNA in polyacrylamide gel via post gel staining. Precast polyacrylamide gel staining with GelRed is not recommended because of relatively high background fluorescence

11、.GelRed Nucleic Acid Gel Stain 10,000X in DMSO is a concentrated Gelgreen solution that can be diluted 10,000 times for use in precast gel staining for 3,300 times for use in post gel staining according to the procedures described below. One vial (0.5ml) of 10,000X solution can be used to prepare at

12、 100 precast minigels or post-stain at least 100 minigels.Gel staining with GelRed is compatible with downstream applications such as gel extraction and cloning. GelRed is efficiently removed from DNA by phenol/chloroform extraction and ethanol precipitation.FeaturesØ Safety: Nonmutagenic and n

13、oncytotoxicØ Easy disposal: Safe to dispose in the drainØ Compatibility: Spectrally compatible with existing instrumentsØ Sensitivity: Higher signal but lower backgroundØ Stability: can be stored at RT and microwavable1.Post-staining Protocol1.1 Run gels as usual according to you

14、r standard protocol.1.2 Dilute the GelRed 10,000X stock reagent3,300 fold to make a 3X staining solution in H2O with 0.1M Nacl(e.g, add 15ul of GelRed 10,000X stock reagent and 5ml 1M Nacl to 45ml H2O). Note: including 0.1M NaCl in the staining solution enhances sensitivity, but may promote dye prec

15、ipitation if the gel stain is reused.1.3 Carefully place the gel in a suitable polypropylene container. Gently add a sufficient amount of the 3X staining solution to submerge the gel.1.4 Agitate the gel gently at room temperature for30 minutes.1.5 Image the stained gel with a standard transilluminat

16、or (302 or 312nm), and photograph the gel using an ethidium bromide filter.1.6 Staining solution can be reused at least 23times. Store staining solution at room temperature protected from light.2. Pre-cast Protocol2.1 Prepare molten agarose gel solution using your standard protocol.2.2 Dilute the Ge

17、lRed 10,000X stock reagent into the molten agarose gel solution at 1:10,000 (e.g.,5ul of GelRed 10,000Xstock reagent added to 50ml of the gel solution)and mix thoroughly. Gelgreen can be added while the gel solution is still hot.2.3 Cast the gel and allow it to solidify. Any leftover gel solution ma

18、y be stored and reheated later for additional gel casting. GelRed precast gels may be stored at 4°C for later use.2.4 Load samples and run the gels using your standard protocol.2.5 Image the stained gel with a standard transilluminator(302 or 312nm), and photograph the gel using an Ethidium bro

19、mide filter.Note: The pre-cast protocol is not recommended for polyacrylamide gels. Although the post-staining method is recommended, precast gels may also be tried with GelRed. However, some DNA samples, such as those derived from plasmid DNA digestion by certain restriction enzymes, may experience

20、 migration retardation or compromised resolution. Thus, both the post-stained and precast gels can be performed to determine which one may better meet your needs. Figure 2. GelRed Pre-cast ProtocolFigure 1. Normalized excitation and emission spectra of Gelgreen (green) and GelRed (red) in the presen

21、ce of dsDNA in PBS bufferGelRed and Gelgreen troubleshooting1. Why am I seeing smeared or smiling DNA band(s) or discrepant DNA migration? GelRed and Gelgreen cannot penetrate live cell membranes then go into the DNA double helix structure like EB because they are larger mol

22、ecules (big molecules than EB). They are high affinity dyes designed to be larger dyes to improve their safety. So they may affect the migration of DNA in precast gels. Specially, such as restriction digested DNA may migrate abnormally in precast gels.Please try the following methods to re

23、duce the smeared or smiling DNA band(s) or discrepant DNA migration:1) Tip #1: Load less DNA Smearing and smiling in GelRed or Gelgreen precast gels most often caused by overloading of DNA. If you see band migration shifts or smearing and smiling, try reducing the amount of DNA loaded. The recommend

24、ed loading amount for ladders and samples of known concentration is 50-200 ng/lane. For samples of unknown concentration, try loading one half or one third of the usual amount of DNA. This usually solves band migration problems. Blown out or smeared bands can be caused by overloading. This is freque

25、ntly observed with DNA ladders. 2) Tip #2: Try the post-staining protocol To avoid any interference the dye may have on DNA migration, we recommend using the post-staining protocol. If your application requires loading more than the recommended amount of DNA, use the post-staining protocol. While we recommend post-staining gels for 30 minutes, you may be able see bands in as little as five minutes, depending on how much DNA is present. Post-staining solutions can be reused.3) Tip #3: Pour a lower percentage agarose gel for better resolution of la