土壤试剂盒操作手册和常见问题

FastDNA Spin Kit for Soil实验步骤1.Add up to 500 mg of soil sample to a Lysing Matrix E tube.在裂解介质管E中最多加入500mg土壤样品。

注意：推荐最多加入500mg土壤样品，含水量比较多的土壤或者碎屑多的土壤可适量减少样品量。

2.Add 978 μL Solution Phosphate Buffer to sample in Lysing Matrix E tube.在裂解介质管E中加入978μl Sodium Phosphate Buffer3.Add 122 μL MT Buffer.What’s happening: Begin to solubilize membrane proteins with detergents as well asextra-cellular proteins and contaminations in soil.加入122μl MT Buffer发生的反应：用洗涤剂溶解细胞膜蛋白以及细胞外蛋白和土壤中的污染物。

注意：为了得到更好的样品处理效果，加入土壤样品及两个缓冲液后，在裂解介质管中仍能保留有250-500μl空间。

4.Homogenize in the FastPrep Instrument for 40 seconds at a speed setting of 6.0What’s happening: mechanical disruption of cell walls of soil organisms and releasing nucleic acids into the protective buffer.将样品置于FastPrep®仪器上匀浆40s，速度为6.0m/s发生的反应：机械破碎土壤微生物的细胞壁，将核酸释放入保护缓冲液中。

5.Centrifuge at 14,000×g for 5-10 minutes to pellet debris.14,000 x g离心5-10min至沉渣注意：如果把离心时间延长到15min，可以更好地使样品量较大的，或者细胞壁结构较复杂的细胞碎片沉降到管底。

6.Transfer supernatant to a clean 2.0 ml microcentrifuge tube. Add 250μL PPS (Protein PrecipitationSolution) and mix by inverting the tube 10 times.What's happening: Separate the solubilized nucleic acids from the cellular debris andlysing matrix. Flocculation of protein-containing micelles 将上清液转移至一个干净的2.0ml离心管中。

加入250μL的PPS溶液（蛋白质沉淀溶液），用手颠倒10次，使之充分混合。

发生的反应：将溶解的核酸与细胞沉渣以及裂解介质分离。

产生絮状蛋白。

7.Centrifuge at 14,000 x g for 5 minutes to pellet precipitate. Transfer supernatant to a clean 15mltube.What's happening: Removal of flocculated proteins.14,000 x g离心5min至沉淀。

将上清液转移至一个干净的15ml管中发生的反应：去除絮状蛋白。

注意：此步也可使用2.0ml离心管，但使用大管可以更好的混匀以及DNA的结合。

8.Resuspend Binding Matrix suspension and add l.0 ml to supernatant in 15ml tube.重悬Binding Matrix溶液，将1.0 ml重悬液加入该15ml管中。

9.Place on rotator or invert by hand for 2 minutes to allow binding of DNA. Place tube in a rack for3 minutes to allow settling of silica matrix.What’s happening: Nucleic acid bind to the silica matrix in the presence of chaotropic salts.用摇床或手动颠倒混匀2min，使DNA更好的与binding matrix结合。

之后将管子置于管架上，静置3分钟，使binding matrix自然沉降。

发生的反应：在离液盐存在的情况下，核酸与binding matrix结合。

10.Remove and discard 500 μL of supernatant being careful to avoid settled Binding Matrix.小心地去除500μl上清液，避免吸到沉淀下来的binding matrix。

11.Gently resuspend Binding Matrix in the remaining amount of supernatant. Transfer approximately600 μL of the mixture to a Spin TM Filter and centrifuge at 14,000 x g for 1 minute. Empty the catch tube and add the remaining mixture to the Spin Filter and centrifuge as before. Empty the catch tube again.用剩余的上清液轻轻的重悬binding matrix，转移大约600μl的重悬液至SPIN™ Filter中，14,000 x g离心1min，弃去收集管中的废液。

将15ml管中剩余的重悬液转移到SPIN™ Filter中，再次离心弃去废液。

12.Add 500μL prepared SEWS-M and gently resuspend the pellet using the force of the liquid fromthe pipet tip.What 's happening: Continuing to solubilize proteins.加入500μl预先准备好的SEWS-M溶液，用枪头轻轻吹打，小心地重悬沉淀。

发生的反应：继续溶解蛋白。

注意：使用前确保SEWS-M溶液中已加入乙醇。

在SEWS-M溶液中加入100ml 100%的乙醇，混匀。

在瓶子上做好标记，密闭储存于室温。

13.Centrifuge at 14,000 x g for l minute. Empty the catch tube and replace.What's happening: Desalting with Ethanol and additional detergents remove impurities bycentrifuging through the Spin filter bucket while the purified DNA is still boundto the silica.14,000 x g离心1min，弃去废液。

发生的反应：通过离心过滤，用脱盐的乙醇和去垢剂去除杂质，而DNA仍然与binding matrix 结合。

14.Without any addition of liquid, centrifuge a second time at 14,000 x g for 2 minutes to "dry" thematrix of residual wash solution. Discard the catch tube and replace with a new, clean catch tube.不加任何溶液，将SPIN™ Filter 14,000 x g离心2min，去除残留的SEWS-M溶液。

弃去收集管，更换一个新的、干净的离心管。

15.Air dry the Spin TM Filter for 5 minutes at room temperature.What's happening: Removal of residual ethanol.将SPIN™ Filter置于室温，晾干5min。

发生的反应：去除残留的乙醇。

16.Gently resuspend Binding Matrix (above the Spin fitter) in 50-100uL of DES (DNase/Pyrogen-Free Water).What's happening: Purified nucleic acids elutes from silica with collapse of cation bridge because low salt elution solution rehydrates both the silica and the DNA.在SPIN™ Filte r中加入50-100μl的DES溶液（或者无菌/无DNA酶的水），轻轻的重悬binding matrix。

发生的反应：低盐洗脱液使binding matrix和DNA再水合化，导致盐桥断裂，从而使纯化的核酸从binding matrix中洗脱下来。

注意：a：为了避免过度稀释纯化出的DNA，请尽量减少DES溶液的量b：55˚C水浴孵育5min能提高纯化产物的得率17.Centrifuge at 14,000 x g for l minute to bring eluted DNA into the clean catch tube. Discard theSpin filter, DNA is now ready for PCR and other downstream applications. Store at -20℃for extended periods or 4aC until use.What's happening: Final purified DNA passes through filter bucket and is collected in a cleanmicrofuge tube.14,000 x g离心1min，使洗脱的DNA转移至收集管中。