1.Weigh 500 mg of glass beads in a 15 ml centrifuge tube, add 0.2-0.5 g soil sample. Add 1 ml Buffer SLX Mlus. Vortex at maximum speed for 3-5 minute to lyse sample. For the best result, A Mixer Mill,such as Fastprep-24,Mixer Mill 300, should be used.
稱取500毫克的玻璃珠在15毫升離心管,添加0.2 -0.5 g土壤樣品。加入1毫升SLX Mlus緩沖液。最大速度下攪拌3 - 5分鐘直到樣品溶解。為達(dá)到最佳效果,需要用到攪拌磨,如Fastprep-24、Mixer Mill 300。
2.Add 100 ul Buffer DS and vortex to mix.
加入100ulDS緩沖液，攪拌混勻。
3.Incubate at 70 ℃ for 10 minute. Briefly vortex the tube once during the incubation. For some difficult lysis bacterial. Increase the temperature to 90 ℃.
在70 ℃ 培養(yǎng)10分鐘。在培養(yǎng)的過程中稍微地?cái)嚢桦x心管一次，對(duì)一些比較難溶解的細(xì)菌，將溫度調(diào)至90℃。
4.Centrifuge at 3000 rpm for 3 min at room temperature. Transfer 800 ul the supernatant into a new 2 ml tube and add 270 ul Buffer SP2.
Mix the sample throughly by vortexing.室溫下3000rpm離心3分鐘。轉(zhuǎn)移800ul上清液到一個(gè)新的2 ml 管，然后加入270 ul SP2 緩沖液。攪拌，徹底地混勻樣品。
5.Incubate in ice for 5 min. Centrifuge at full speed (13,000 ×g) in a microcentrifuge for 5 min at 4 ℃.
冰上孵育5分鐘。在微離心機(jī)下，4℃全速（13,000 g）離心5分鐘。
6.Carefully transfer supernatant to a new 2 ml tube and add 0.7 volume of isopropanol. Mix throughly by inverting tube for 20 -30 times. If the soil contains very low DNA, incubate the sample at -20℃ for 1 hour.
小心地轉(zhuǎn)移上清液到一個(gè)新的 2 ml 管，然后加0.7體積的異丙醇。通過反向管徹底地混合20-30次。假如土壤中含少量的DNA，需要在-20℃ 孵育1h。
7.Precipitate DNA by centrifuge at full speed (13,000 ×g) for 10 minute at 4 ℃ .
4 ℃下全速(13,000 ×g)離心10分鐘，沉淀DNA。
8.Carefully discard the supernatant and make sure not dislodge the DNA pellet. Invert the tube on a absorbent paper for 1 minute to drain the liquid. It is not necessary to dry the DNA pellet.小心地棄掉上清液，確保不攪動(dòng)DNA沉淀。倒置離心管在吸水紙上1 分鐘，使液體流盡。不需要干燥DNA沉淀。
9.Add 200 ul of Elution Buffer to the tube and vortex for 10 seconds.Incubate at 65℃ for 10- 20 minutes to dissolve the DNA pellet.加 200 ul 洗脫緩沖液，然后攪拌10秒鐘。在 65℃ 孵育10-20 分鐘，溶解DNA 沉淀。
10.Add 50 -100 ul of HTR Reagent. Mix throughly by vortexing for 10 minute. Note :completely resuspend HRT Reagent by shaking the bottle before use.
加 50- 100ul HTR 試劑，攪拌10分鐘，徹底混勻。注意：用前搖動(dòng)瓶子，充分懸浮HTR 試劑。
11.Incubate at room temperature for 2 minutes. Centrifuge at full speed (13,000 ×g) for 2 minutes.室溫下孵育2分鐘。全速 (13,000 ×g)離心2分鐘。
12.Transfer cleared supernatant to a new2 ml tube.Note :if supernatant still have dark color from soil ,perform the HTR treatment again by repeat step 10-12.
轉(zhuǎn)移清澈的上清液到一個(gè)新的2ml管。注意：假如上清液呈黑色，重復(fù)10-12步。
13. Add equal volume of XP2 Buffer ,mix by vortexing. For examper: if the sample from step 12 is 250 ul, add 250 ul Buffer XP2.
加入等量的XP2 緩沖液，攪動(dòng)混勻。如：12步后的量是250ul，就加入250ul的XP2緩沖液。
14.Apply the sample from step 13 to a HiBind DNA Column assembled in a 2 ml collection tube(supplied).centrifuge at 10,000×g for 1 minute at room temperature. Discard flow-through liquid and re-use collection tube.
將13步的樣加到組裝于2ml收集管（已提供）的HiBind DNA Column。室溫下10,000×g 離心1分鐘，倒掉直流液體，再次使用收集管。
15.Place the column into the same 2 ml collection tube from previous step and add 300 ul XP2 Buffer. Centrifuge at 10,000×g for 1 minute. Discard the flow-through and collection tube.將柱子放到相同的先前的2ml收集管，然后加入3000ulXP2緩沖液。10,000×g離心1分鐘。棄掉液體和收集管。
16.Place column into a new 2 ml collection tube(supplied) and wash by adding 700 ul SPW Wash Buffer diluted with absolute ethanol. Centrifuge at 10,000×g for 1 minute.Discard flow-through liquid and re-use collection tube in next step.Note:SPW Wash Buffer is provided as a concentrate and must bediluted with absolute ethanol as indicated on the bottle and page 4.
將柱子置于一個(gè)新的2ml收集管（已提供），加入700ul 用無水乙醇稀釋的SPW洗脫液洗脫。10,000×g離心1分鐘。棄掉直流液，收集管下一步再次使用。注意：SPW洗脫液必須濃縮，必須用無水乙醇再次稀釋，此項(xiàng)注明在瓶子上和第4頁。
17.Repeat step 16 with a second 700 ul SPW Wash Buffer.
重復(fù)16步。
18.Discard liquid and re-insert the column to the empty collection tube,centrifuge the column at full speed(13,000 ×g) for 2 minutes at room temperature. This step is critical in removing traces of ethanol that will interfere with downstream applications.
棄掉液體，重新插入柱子到空的收集管，室溫下全速離心2分鐘。這步對(duì)清除乙醇痕跡很重要。因?yàn)橐掖紩?huì)干擾下面步驟的應(yīng)用。
19.Place column into a clean 1.5 ml microcentrifuge tube (not supplied). Add 30- 100 ul Elution Buffer directly onto the centre of HiBind matrix. Incubate at 65 ℃ for 10-15 minutes.
將柱子置于一個(gè)干凈的1.5ml的微離心機(jī)管（沒有提供）。直接加入30-100ul洗脫液在HiBind matrix的中心。在65 ℃下孵育10-15分鐘。
20.Centrifuge at full speed (13,000 ×g) for 1 min to Elute DNA.
全速離心1分鐘，洗提DNA。
21.Repeat elution step 19-20 with a second 30-100 ul Elution Buffer.
重復(fù)洗脫步驟19-20。
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