试剂盒提取详细步骤.docx

1、试剂盒提取详细步骤美国MOBIO土壤RNA提取试剂盒(RNA PowerSoil Total RNA Isolation KitDetailed Protocol (Describes what is happening at each stepb5E2RGbCAPWear RNase-Free gloves (1555 at all times and remove RNase from the work area usingLab Cleaner (12095 for RNase Removal. Both of these products are available from MOBI

2、O. Please see “Other Quality Products Available” section at the end of this manual.p1EanqFDPw1. Add up to 2 g of soil to the 15 ml Bead Tube (provided. Note: Please refer to Hints andTroubleshooting Guide for information regarding the amount of soil to process.DXDiTa9E3d1、加2g土壤到15ml磁珠管中。注意：请参照提示和纠错指

3、南确定加入土壤的量。2. Add 2.5 ml of Bead Solution to the Bead Tube and vortex to mix.RTCrpUDGiT2、加2.5mlBead Solution到磁珠管中并漩涡混合。3. Add 0.25 ml of Solution SR1 to the Bead Tube and vortex to mix.5PCzVD7HxAWhats happening: The Bead Solution is a buffer used to disperse cells and soil particles. Solution SR1cont

4、ains SDS and other disruption agents which aid in complete cell lysis. In addition to aiding in cell lysis,SDS is an anionic detergent that breaks down fatty acids and lipids associated with the cell membrane ofseveral organisms. Note: If it gets cold, it will form a white precipitate. Heating to 60

5、C will dissolve theSDS and will not harm the other disruption agents.jLBHrnAILg3、加0.25ml SR1到磁珠管并漩涡混合。发生的反应：Bead Solution是一种缓冲液可用来打散细胞和土壤颗粒。SR1 包括SDS和其他的能帮助细胞裂解的破碎剂。除了帮助细胞破碎外，SDS是阴离子洗涤剂，可以破坏脂肪酸和几种微生物细胞膜相关的脂类。xHAQX74J0X注意：如果天气冷了，可能会形成白色沉淀。加热到60溶解SDS，且不会影响其他裂解试剂的作用。4. Add 0.8 ml of Solution SR2 and pl

6、ace the Bead Tube on the Vortex Adapter (MO BIO Catalog #13000-V1-15 for Vortex Genie 2 or 13000-LV2-15 for Labnet Vortex and vortex at maximumspeed for 5 minutes.LDAYtRyKfEWhats happening: Solution SR2 is a precipitation reagent used to remove non-DNA organic and inorganicmaterial including humic s

7、ubstances, cell debris, and proteins. Contaminating organic and inorganic mattermay reduce DNA purity and inhibit downstream DNA applications. Vortexing is critical for homogenizationand cell lysis.Zzz6ZB2Ltk4、加0.8ml SR2到磁珠管，最大转速旋转5min。反应：SR2是一种沉淀试剂，用来去除非DNA有机物和无机物包括腐殖质，细胞碎片以及蛋白质。污染有机物和无机物质可能会降低DNA纯

8、度和抑制后续的DNA利用。漩涡对细胞均化和细胞裂解至关重要。dvzfvkwMI15. Remove the Bead Tube from the Vortex Adapter and add 3.5 ml of phenol: chloroform:isoamylalcohol (pH 6.5 8.0, User supplied and vortex to mix until the biphasic layer disappears.rqyn14ZNXI5、加3.5ml酚/氯仿/异戊醇 to a clean 15 ml Collection Tube (provided.The thick

9、ness of the interphase will vary depending on the type of soil used. Discard thephenol:chloroform:isoamyl alcohol in an approved waste receptacle. Note: The biphasic layerwill be thick and firm in soils high in organic matter and may need to be pierced to remove thebottom phenol layer for disposal.s

10、QsAEJkW5T8、小心地移取上层水相到一15ml收集管中。注意：有机物含量高两相层可能会较厚较坚固，可能需要移取底层的酚相来处理。Whats happening: The upper aqueous phase containing the total nucleic acids from the sample istransferred to a new tube. Cellular debris, proteins, and organic material are left behind. Note: Take carenot to transfer material from th

11、e lower phase or the interphase.GMsIasNXkA反应：包括样品总核酸上层水相转移到新收集管中。丢弃细胞碎片，蛋白质和有机物。注意：不要碰到中间相和下层酚相。9. Add 1.5 ml of Solution SR3 to the aqueous phase and vortex to mix. Incubate at 4C for 10minutes.TIrRGchYzgWhats happening: Solution SR3 is a secondary precipitation step to further remove proteins and

12、cellulardebris.7EqZcWLZNX9、加1.5mlSR3到水相中，漩涡混合。4静置10min。反应：加SR3是二次沉淀步骤，进一步去除其中的蛋白质和细胞碎片。10. Centrifuge at 2500 x g for 10 minutes at room temperature.lzq7IGf02E10、室温2500g离心10min。11. Transfer the supernatant, without disturbing the pellet, to a new 15 ml Collection Tube(provided.zvpgeqJ1hkWhats happen

13、ing: The supernatant containing nucleic acids are transferred to a new 15 ml tube. Nonnucleicacid material is left behind.NrpoJac3v111、将上清液转移到15ml收集管中mZkklkzaaPWhats happening: Solution SR5 is a proprietary salt solution used to resuspend the precipitated nucleicacids from step 14. It is also used t

14、o equilibrate the RNA capture column in step 16 and to wash and prepthe column for the elution of RNA in step 20 below.AVktR43bpw15、摇晃SR5使其混合，加1mlSR5到收集管中，使沉淀再完全悬浮。注意：土壤样品不同，沉淀有可能不易悬浮，可能需要将收集管放到45的水浴池中10min再悬浮,再漩涡混合，重复这样直到沉淀重悬浮。ORjBnOwcEd16. Prepare one RNA Capture Column (provided for each RNA Isol

15、ation Sample:2MiJTy0dTTa. Remove the cap of a 15 ml Collection Tube (provided and place the RNA CaptureColumn inside the 15 ml Collection Tube. The column will hang in the 15 ml CollectionTube.gIiSpiue7Ab. Add 2 ml of Solution SR5 to the RNA Capture Column and allow it to gravity flowuEh0U1Yfmhthrou

16、gh the column and collect in the 15 ml Collection Tube. Allow Solution SR5 toIAg9qLsgBXcompletely flow through the column (Optional: The Collection Tube may be emptied afterSolution SR5 has completely flowed through the column. Note: DO NOT ALLOW THECOLUMN TO DRY OUT PRIOR TO LOADING THE RNA ISOLATI

17、ON SAMPLE.WwghWvVhPE16、为每个RNA样品准备一个捕集柱。a.将捕集柱悬挂到收集管上。b. 加2mlSR5到捕集柱上，使其重力流。允许SR5完全流过捕集柱。注意：在加RNA样品前不要让捕集柱流干。17. Add the RNA Isolation Sample from Step 15 onto the RNA Capture Column and allow it togravity flow through the column. Collect the flow through in the 15 ml Collection Tube.asfpsfpi4k17、将15

18、步的RNA分离样加到捕集柱中，使其流过捕集柱。18. Wash the column with 1 ml of Solution SR5. Allow it to gravity flow and collect the flow through inthe 15 ml Collection Tube.ooeyYZTjj1Whats happening: The sample is added to the RNA Capture Column and the nucleic acids are bound tothe column matrix. The Capture Column is

19、then washed with a second volume of Solution SR5 to ensureunbound contaminants are removed from the sample and column prior to the elution of RNA.BkeGuInkxI18、用1mlSR5洗涤捕集柱，流出液收集在收集管中。反应：样品加到捕集柱上，核酸结合到柱基质上。捕集柱用SR5洗涤确保没结合的污染物被去除掉。19. Transfer the RNA Capture Column to a new 15 ml Collection Tube (prov

20、ided. Shake SolutionSR6 and then add 1 ml of Solution SR6 to the RNA Capture Column to elute the bound RNAinto the 15 ml Collection Tube. Allow Solution SR6 to gravity flow into the 15 ml CollectionTube.PgdO0sRlMoWhats happening: The Solution SR6 RNA elution buffer is a proprietary salt solution tha

21、t allows for thepreferential release of RNA from the RNA Capture Column leaving DNA, residual debris, and inhibitingsubstances in the column. Note: a new kit is available for DNA elution. See DNA Elution Procedure inthe Hints and Troubleshooting Guide or contact MO BIO for details at technical3cdXwc

22、km1519、将捕集柱转移到新收集管中，摇晃SR6，然后加1mlSR6到捕集柱中使其流过捕集柱，洗提RNA。反应：SR6 RNA洗提缓冲液是专有的盐溶液，它能使RNA流出而DNA、剩下的细胞碎片和抑制剂依然留在捕集柱上。h8c52WOngM20. Transfer the eluted RNA to a 2.2 ml Collection Tube (provided and add 1 ml of Solution SR4.Invert at least once to mix and incubate at -20C for 10 minutes.v4bdyGious20、将洗提的RNA转移到2.2ml的收集管中，并加1mlSR4，至少倒置混合一次，-20静置10min。J0bm4qMpJ921. Centrifuge the 2.2 ml Collection Tube at 13,000 x g for 15 minutes at room temperature to