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 Buffer

3.Add 122 μL MT Buffer.

What’s happening: Begin to solubilize membrane proteins with detergents as well as

extra-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 6.0

What’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 Precipitation

Solution) and mix by inverting the tube 10 times.

What's happening: Separate the solubilized nucleic acids from the cellular debris and

lysing 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 15ml

tube.

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 for

3 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 approximately

600 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 from

the 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 by

centrifuging through the Spin filter bucket while the purified DNA is still bound

to 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" the

matrix 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 the

Spin 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 clean

microfuge tube.

14,000 x g离心1min，使洗脱的DNA转移至收集管中。弃去SPIN? Filter。得到的DNA纯化产物可直接用于PCR及其它下游实验。使用前可储存于4℃，长期保存可置于-20℃。

发生的反应：最后纯化的DNA可通过过滤柱，收集至一个新的离心管中。

FastDNA Spin Kit for Soil常见问题

1.Wet Soil Sample

土壤样品过湿

If the soil is extremely wet, transfer the Lysing Matrix E components to another sterile holding tube. Place the soil sample in the empty matrix tube, and centrifuge 30 seconds at 10,000 x g. Decant as much liquid as possible, replace lysing matrix components and continue with protocol.

如果土壤样品过湿，可将裂解介质管E中的内容物转移至一个新的无菌管中。将土壤样品加入到空的裂解介质管中，10，000 x g离心30s，尽可能多的去除液体，之后再加入裂解介管的内容物，继续下面的实验步骤。

2.Low DNA Yield in Eluate

DNA产量低

Insufficient Lysis-While a FastPrep speed setting of 6.0 m/sec and a 40 second run time will be adequate for most soil types, additional processing may be necessary. If repeat processing cycles are necessary it is recommended to incubate Lysing Matrix tubes in ice for 2 minutes between cycles to avoid excessive heat build up.

裂解不充分：大部分土壤样品在速度6m/s,时间40s的条件下可充分裂解，但是少部分样本需要额外的裂解。如果有必要重复匀浆裂解，建议在两个循环之间，将裂解介质管置于冰上2min，以避免过热。

Insufficient Binding Matrix-Binding matrix is supplied as a suspension and must be completely dispersed before pipetting. Vigorously shake the bottle of binding matrix to produce a uniform suspension. In some instances, vortexing may be necessary.

Binding Matrix的使用量不足：Binding Matrix是悬浮液，在使用前需要用力摇匀，必要时可涡旋震荡。

Ethanol not added to SEWS-M solution-SEWS-M solution is supplied as a concentrate. 100 ml of 100% Ethanol must be added to the concentrate before use.

SEWS-M中没有加入乙醇：使用前需要加入100ml100%乙醇。

DNA not eluted efficiently-To increase elution efficiency, after resuspending binding matrix with DES solution, incubate for 5 minutes at 55℃before centrifuging the final eluate.

洗脱不充分：为了增加洗脱效率，用DES溶液重悬binding matrix后，在最终的洗脱离心前，可在55℃孵育5min。

3.DNA does not amplify

DNA无法扩增

Quantitate DNA Yield-by running gel, or spectrophometer. Excess DNA will inhibit PCR reactions.

检测DNA的浓度：通过电泳或者分光光度计测定，过量的DNA模板也会抑制PCR反应。

Dilute Template DNA-This should not be necessary with DNA isolated with the FastDNA Spin Kit for Soil, but is still an option.

稀释模板DNA：这一步不是必须的，但是可以选择。

Verify PCR Optimization Conditions-Changing reaction conditions or primer selection may be necessary.

确定PCR反应条件是否已优化：改变反应条件或者引物的选择。

Non-specific bands-Check possibility that target DNA is in low abundance in the eluate.1t is possible that some species of interest, particularly parasitic cysts and oocytes may need additional processing or even more aggressive lysing matrix (such as Lysing Matrix A) in order to disrupt the thick protein ceil wall.

非特异条带：洗脱液中目的DNA的丰度可能比较低。在样品中感兴趣的微生物种类，尤其是寄生虫包囊以及卵子，为了破碎比较厚的蛋白细胞壁，可能需要额外的匀浆裂解，甚至是需要使用更强的裂解介质（例如裂解介质A）。

4.DNA Fragmented

DNA片段化

Use Care with Liquid Transfer-Once the DNA is bound to the binding matrix, care should be taken to avoid DNA shearing. All liquid manipulations, especially resuspension of matrix in SEWS-M solution and DES solution should be performed gently and deliberately. The use of wide-bore pipet tips is recommended for these steps.

小心的转移液体：一旦DNA与binding matrix结合，一定要注意避免DNA断裂。所有的液体操作步骤，尤其是用SEWS-M溶液和DES溶液重悬binding matrix一定要轻柔操作。这些步骤推荐使用宽口的tips。

Optimize Lysis Conditions-High powered bead beating cell disrupters can shear DNA if process settings are too long or powerful, While FastPrep speed setting of 6.0 m/sec and a 40 second run time will be adequate for most soil types, it is possible that lowering speed and/or duration settings will result in higher MW DNA.

优化裂解条件：FastPrep仪器设置速度6.0m/s，时间40s对于大多数土壤样品来说已经足够，低速或者短时可能得到高分子量的DNA。FastPrep仪器设置的速度过大或者时间过长可能导致DNA断裂。

5.Low A260/A280 Ratios for purified DNA

A260/A280比值偏低

Ethanol not added to SEWS-M solution-SEWS-M solution is supplied as a concentrate. 100 ml

of 100% Ethanol must be added to the concentrate before use.

SEWS-M中未加入乙醇：试剂盒提供的SEWS-M溶液是浓缩液，在使用之前需要加入100ml 无水乙醇。

Proteins not removed efficiently-PPS solution must be efficiently mixed in the lysate (Step 6.) Invert tube by hand at least 10 times, or mix by pipet pumping. A 5 minute incubation on ice can further precipitate proteins from difficult samples.

蛋白去除不干净：PPS溶液必须与裂解液充分混匀（操作步骤第6步）。用手颠倒混匀至少10次，或者用移液器轻轻吹打。也可在冰上放置5min，可以增加蛋白的沉淀率。

Contaminants not removed efficiently-During the wash with SEWS-M solution (Step 12), it is necessary to resuspend the entire binding matrix pellet to efficiently remove contaminants. This step should be performed gently and deliberately. The use of wide-bore pipet tips is recommended for these steps.

杂质去除不干净：在操作步骤第12步的洗涤过程中，要充分重悬binding matrix。这一步必须轻柔操作，也可使用宽口的tips。

6.Elevated A230 absorbance

提高A230的吸收值

Proteins not removed efficiently-PPS solution must be efficiently mixed in the lysate (Step 6.) Invert tube by hand at least 10 times, or mix by pipet pumping. A 5 minute incubation on ice can further precipitate proteins from difficult samples. For samples suspected to contain very high levels of inhibitors, an additional PPS treatment can be done.

蛋白去除不干净：PPS溶液必须与裂解液充分混匀（操作步骤第6步）。用手颠倒混匀至少10次，或者用移液器轻轻吹打。也可在冰上放置5min，可以增加蛋白的沉淀率。对于抑制物含量比较高的样品，可重复PPS溶液沉淀蛋白的步骤。

Contaminants not removed efficiently-During the wash with SEWS-M solution (Step 12), it is necessary to resuspend the entire binding matrix pellet to efficiently remove contaminants. This step should be performed gently and deliberately. The use of wide-bore pipet tips is recommended for these steps. For samples suspected to contain very high levels of inhibitors, an additional PPS treatment can be done.

杂质去除不干净：在操作步骤第12步的洗涤过程中，要充分重悬binding matrix。这一步必须轻柔操作，也可使用宽口的tips。

Residual Ethanol in the final eluate-Be sure to perform additional 2 minute centrifugation step (Step 14) after all of the SEWS-M solution has been run through the column. Performing the 5 minute air dry (Step 15) with a 60℃incubation can also aid in removing residual Ethanol.

最终的洗脱液中乙醇残留：确保SEMS-M溶液通过过滤柱之后，再次离心2min（操作步骤第14步）。5min晾干（操作步骤第15步）以及60℃孵育可帮助去除残留乙醇。