DNeasy® 96 Plant Protocol for Isolation of DNA from Fresh Plant Leaves Using the Mixer Mill MM 300

一、Important notes before starting （使用前的重要注释）

1、Take time to familiarize yourself with the Mixer Mill MM 300 and the Centrifuge 4-15C/ Centrifuge 4K15C before starting this protocol（开始使用本实验方法之前花时间熟悉搅拌磨MM 300和离心机4-15C/ 离心机4K15C）。

2、This protocol describes processing of 192 samples (2×96). If you wish to process 96 or fewer samples, provide a balance for the mixer mill by assembling a second plate sandwich using a rack of collection microtubes without samples or buffers, but containing tungsten carbide beads, and fixing this second sandwich into the empty clamp （本方法用来对2×96=192个样本进行提取DNA ，如果希望处理96个或更少样本，要对搅拌磨MM 300进行平衡处理，方法是用一架没有样本和缓冲液但装有碳化钨珠子的收集微型管插入第二个孔板，并用空夹子加入固定）。

3、Tungsten carbide beads are reusable. See the Appendix for recovery and cleaning details （碳化钨珠子可再利用，详见附录）。

4、Ensure that ethanol has been added to Buffers AP3/E and AW（确保乙醇已加入AP3/E和AW缓冲液中）。

5、Preheat Buffer AP1 to 65℃. This heating is necessary for the DNeasy 96 Plant procedure, and will also dissolve any precipitate that may have formed in Buffer AP1（将AP1缓冲液预热到65℃。这个预热过程对于DNeasy 96 Plant实验技术是必须的。另外，如果AP1缓冲液中形成沉淀，也要将其溶解）。

6、All centrifugation steps should be performed at room temperature. If a Centrifuge 4K15C is used, set the temperature to 40℃ for all centrifugation steps（所有离心步骤都应在室温下进行。如果使用4K15C型离心机，要将所有离心步骤的温度调至40℃）。

7、Wear safety glasses throughout the procedure（全过程都要戴安全镜）。

8、IMPORTANT: DNA can appear as a smear on agarose gels when the liquid grinding protocol is used. This can be avoided by using the frozen grinding protocol（特别重要的是：使用液体研磨方法，DNA 会在琼脂糖凝胶上出现模糊现象，而用冰冻研磨法则可避免）。

二、（Procedure）实验步骤

1 、Harvest leaves and place up to 50 mg into each tube in two collection microtube racks （收集叶片，并在两个微型管架上的每个管中加入50 mg 叶片）。

Unless the optimal amount of starting material has been determined, do not use more than 50 mg (wet weight) per sample（除非已经确定开始材料的最适数量，否则每个样本不要使用超过鲜重50 mg）。

Most leaf material can be stored at 4℃ for at least 24 h prior to processing without affecting DNA yield or quality（处理前将大部分叶片材料在4℃贮藏至少24 h不影响DNA 的产量和质量）。

Keep the clear covers from the collection microtube racks for use in step 7（在步骤7中使用的微型收集管架盖子要保持清洁）。

Use the provided plate register cards to record the position of each sample in the racks（使用提供的微孔板记录卡记录架子中每一个样本的位置）。

3 、Combine Buffer AP1, RNase A, and Regent DX according to the table below to make a working lysis solution. Pipet 400 µl of the working lysis solution into each collection microtube. Seal the microtubes with the provided caps （按照下表将AP1 缓冲液、 RNase A 及试剂DX 混合配成裂解工作液。吸取裂解工作液在每个 微型收集管 加入400 µl 。用提供的盖子将 微型收集管封好 ）。

It is important to prepare fresh working lysis solution. To allow thorough mixing of the solution, combine the components in a tube and vortex to mix, then dispense the solution into a reagent reservoir for use with the multichannel pipet（配制新鲜的裂解工作液是很重要的。为了使溶液充分混匀，将各成分加入管中后，用涡流搅拌器搅拌，然后分装到试剂贮瓶中以利于多级吸量管使用）。

注：15% excess mixture is included in these calculations to allow for pipetting errors（本计算中已包括15%的过量混合液，可不计吸量误差）。

Reagent DX is a viscous solution（试剂DX是一种粘稠液体）。

4 、Disrupt the samples using the Mixer Mill MM 300 as fillows （使用搅拌磨MM 300 裂解样品方法如下）:

a. Sandwich each rack of collection microtubes between adapter plates and fix into the mixer mill clamps as described in the Mixer Mill MM 300 instruction manual （如《拌磨MM 300 使用手册》描述的那样，将每个微型收集管架插入到两个适配微孔板中，并用搅拌磨夹加以固定）

Note: Ensure that the microtubes are properly sealed with caps（注意：确保收集微型管盖封好）。

IMPORTANT: Two plate sandwiches must be clamped to the mixer mill to provide balance. To process 96 samples or less, assemble a second plate sandwich using a rack of collection microtubes containing tungsten carbide beads but no samples or buffers, and fix it into the empty clamp（重要的是：插入微型收集管架的两个适配微孔板必须固定到搅拌磨上，以提供平衡。如果希望处理96个或更少样品，要对搅拌磨MM 300进行平衡处理，方法是用另一没有样品和缓冲液但装有碳化钨珠子的收集微型管与两个适配微孔板装配，并用空夹子加入固定）。

b. Shake for 1.5 min at 30 Hz （以30 Hz 摇震1.5 min ）。

IMPORTANT: Prolonging the disruption time may result in shearing of DNA （重要的是：延长裂解时间可能会导致DNA 剪切）。

Rotating the racks of collection microtubes in this way ensures that all samples are thoroughly disrupted. More foam will have formed in the tubes that were outermost during the initial disruption step（用这种方法旋转收集微型管架，以使所有的样品充分裂解。在最初裂解步骤中最外面的管中将会形成更多泡沫）。

IMPORTANT: Merely rotating the entire plate sandwich so that the QIAGEN logos are upside down when reinserted into the mixer mill is not sufficient, since the same samples that were outermost during the initial disruption will remain outermost in the second disruption step（重要的是：当放入搅拌磨时，只是将插入微型收集管架的两个适配微孔板旋转，使QIAGEN商标颠倒是不够的，因为同一样品最初裂解时处于最外面在第二次裂解步骤中也将仍然保持在最外面）。

d. Shake for a further 5 min at 30 Hz （再以30 Hz 摇震1.5 min ）。

IMPORTANT: Prolonging the disruption time may result in shearing of DNA （重要的是：延长裂解时间可能会导致DNA 剪切）。

5 、Remove the plate sandwiches from the mixer mill and remove the adapter plates from each rack of collection microtubes. To collect any solution from the caps, centrifuge the collection microtubes. Allow the centrifuge to reach 3000 rpm, and then stop the centrifuge. Do not prolong this step （将插入微型收集管架的两个适配微孔板取出，并除去适配微孔板。收集盖子里的溶液，将收集微型管进行离心，使转速达到3000 rpm 并停止离心。不要延长这步的时间）。

6 、Remove and discard caps. Add 130 µl Buffer AP2 to each collection microtube （除掉并仍掉盖子，在每一个微型收集管中加入130 µl 缓冲液AP2 ）。

7 、Close the microtubes carefully with new caps (provided); ensure that the microtubes are properly sealed to avoid leakage during shaking. Place a clear cover (saved from step 1) over each rack of collection microtubes and shake the the racks vigorously up and down for 15 s. To collect any solution from the caps, centrifuge the collection microtubes. Allow the centrifuge to reach 3000 rpm, and then stop the centrifuge. Do not prolong this step （用新盖子小心将微型管盖上，确保微型管密封好，不会在震摇过程中泄露。将一个透明盖（自步骤1 留下的）盖在微型收集管架上，上下剧烈震摇15 s 。收集盖子里的溶液，将收集微型管进行离心，使转速达到3000 rpm 并停止离心。不要延长这步的时间）。

Note: To ensure optimal DNA yields, it is important to shake the racks of collection microtubes vigorously up and down with two hands for the full 15 s. The genomic DNA will not be sheared by vigorous shaking（为确保获得最适产量的DNA ，用双手上下剧烈震摇微型收集管架满15秒是很重要的。DNA 将不会被震摇切割）。

The centrifugation step prevents precipitates from freezing to the caps, which would be difficult to remove after incubation at -20℃(step 8)（离心步骤中避免沉淀冷冻在盖子上，因为在-20℃下培养（步骤8）后将难以去除）。

8 、Incubate the racks of collection microtubes for 10 min at -20℃ （将微型收集管架在-20℃ 下培养10 min ）。

This incubation aids the precipitation of proteins and inhibitors of downstream applications following addition of Buffer AP2（培养步骤有助于附加缓冲液AP2后蛋白质和下游应用的抑制物质沉淀）。

9 、Centrifuge the racks of collection microtubes for 5 min at 6000 rpm （ 将微型收集管架在6000 rpm 离心 5 min ）。

Compact pellets will form, but some particles may float. Be careful not to transfer any of these particles in the following step（将会形成致密的沉淀小球，但一些颗粒物质却会浮起来。在以下步骤中注意不要移入这些颗粒物质）。

10 、Remove and discard the caps. Carefully transfer 400 µl of each supernatant to new racks of collection microtubes (provided), ensuring that the new tubes are in the correct orientation. Do not deiscard the pellets as they contain the tungsten carbide beads, which should be recovered (see Appendix) （除去并扔掉盖子。小心每个微型管的上清400 µl 转移至新的微型收集管架，确保新管的方向正确。由于沉淀小球含有碳化钨珠子，不要丢弃沉淀，以后可再收集利用）。

Do not transfer more than 400 µl of the supernatant otherwise the capacity of the DNeasy 96 Plates and the S-Blocks used in subsequent steps will be exceeded（转移的上清不要超过400 µl，否则在以后的步骤中将超出DNeasy 96 Plates和S-Blocks的负载量）。

If less than 400 µl supernatant is recovered, adjust the amount of Buffer AP3/E in step 11 accordingly（如果回收的上清不到400 µl，在第11步中调整AP3/E缓冲液的量）。

Collection microtubes are connected in strips of 8. To avoid transferring particulate matter, it is helpful to remove the strips from the rack so that the contents of the microtubes are visible, and to use the multichannel pipet on its lowest speed setting（微型收集管用8号细条相连，将细条在架上除去，可看到微型管中的内容，从而可避免转移特殊物质。用多级吸量管的最小设置）。

Keep the used collection microtubes to recover the tungsten carbide beads at later stage (see Appendix)（保留用过的微型收集管，在后面的阶段中可回收碳化钨珠子）。

11 、Add 1.5 volumes (typically 600 µl) of Buffer AP3/E to each sample （在每个样品中加入1.5 体积（典型地600 µl ）缓冲液AP3/E ）。

Note: Ensure that ethanol has been added to Buffer AP3/E prior to use（注意：确保缓冲液AP3/E在使用前加入乙醇）。

A white precipitate may form upon addition of Buffer AP3/E. This precipitate does not interfere with the DNeasy 96 Plant procedure or any subsequent application（缓冲液AP3/E加入后会形成白色沉淀。这种沉淀不会干扰DNeasy 96 Plant实验程序及其后的应用）。

12 、Close the microtubes carefully with new caps (provided); ensure that the microtubes are properly sealed to avoid leakage during shaking. Place a clear cover (saved from step 1) over each rack of collection microtubes and shake the the racks vigorously up and down for 15 s. To collect any solution from the caps, centrifuge the collection microtubes. Allow the centrifuge to reach 3000 rpm, and then stop the centrifuge. Do not prolong this step （用新盖子小心将微型管盖上，确保微型管密封好，不会在震摇过程中泄露。将一个透明盖（自步骤1 留下的）盖在微型收集管架上，上下剧烈震摇15 s 。收集盖子里的溶液，将收集微型管进行离心，使转速达到3000 rpm 并停止离心。不要延长这步的时间）。

Note: To ensure optimal DNA yields, it is important to shake the racks of collection microtubes vigorously up and down with two hands for the full 15 s. The genomic DNA will not be sheared by vigorous shaking（为确保获得最适产量的DNA ，用双手上下剧烈震摇微型收集管架满15秒是很重要的。DNA 将不会被震摇切割）。

13 、Place two DNeasy 96 Plates on top of S-Blocks (provided). Mark the DNeasy 96 Plates for later sample identification （将两个DNeasy 96 Plates 置于S-Blocks 上面，作好标记，以利于其后样品鉴定）。

14 、Remove and discard the caps from the collection microtubes. Carefully transfer 1 ml of each sample to the DNeasy 96 Plates （从微型收集管上除去并扔掉盖子。小心将每个样品1 ml 转移到DNeasy 96 Plates ）。

Take care not to wet the rims of the wells to avoid aerosols during centrifugation. Do not apply more than 1 ml（小心操作，不要弄湿微孔边沿，避免在离心过程中喷雾。不要转移超过1 ml的量）。

Note: Lowering pipet tips to the bottoms of the wells may cause sample overflow and cross-contamination. Therefore, remove one set of caps at a time, and begin drawing up the samples as soon as the pipet tips contact the liquid. Repeat until all the samples have been transferred to the DNeasy 96 Plate（注意：将吸量管探入孔底会引起样品漫溢，导致交叉污染。因些一次只移去一套管盖，并且只要吸量管一接触到样品就抽吸样品。重复进行，直到全部样品转移到DNeasy 96 Plate）。

15 、Seal each DNeasy 96 Plate with an AirPore Tape Sheet (provided). Centrifuge for 4 min at 6000 rpm （用AirPore Tape Sheet 密封DNeasy 96 Plate 。在6000 rpm 下离心4 min ）。

AirPore Tape prevents cross-contamination between samples during centrifugation. After centrifugation, check that all of the lysate has passed through the membrane in each well of the DNeasy 96 Plate. If lysate remains in any of the wells, centrifuge for a further 4 min（AirPore Tape可避免在离心过程中样品之间交叉污染。离心完后，检查所有的裂解溶液量否通过DNeasy 96 Plate每个孔底部的滤膜。如果任何孔中仍有残留，再离心4 min）。.

16 、Remove the tape. Carefully add 800 µl Buffer AW to each sample （除去胶纸，小心在每个样品中加入800 µl 缓冲液AW ）。

Note: Ensure that ethanol has been added to Buffer AW prior to use（注意：确保缓冲液AW在使用前已加入乙醇）。

17 、Seal each DNeasy 96 Plate with a new AirPore Tape Sheet (provided). Centrifuge for 15 min at 6000 rpm to dry the DNeasy membranes （用AirPore Tape 密封Sheet DNeasy 96 Plate 。在6000 rpm 下离心15 min 使DNeasy 膜干燥）。

Important: Residual ethanol in the DNeasy membranes derived from Buffer AW may inhibit PCR and must be removed by centrifugation before elution of the DNA （重要的是：自缓冲液AW残留在DNeasy膜上的乙醇会抑制PCR反应，必须在稀释DNA 之前通过离心除去）。

Note: DNeasy membranes are sometimes slightly colored after this wash step. This should not affect the DNeasy 96 Plant procedure. A very dark membrane could indicate that too much starting material was used. A second wash step with 800 µl ethanol (90-100%) may improve DNA quality in these cases. Empty the flow-through from the S-Block before performing this second wash step（DNeasy膜有会时经洗涤步骤之后轻微染色，但这不影响DNeasy 96 Plant实验。如果膜的颜色很暗，说明使用的起始材料太多。这种情况下用800 µl的90-100%乙醇第二次洗涤可提高DNA 质量。在进行第二次洗涤之前可倒空S-Block里面的滤液）。

18 、Remove the tape. To elute the DNA , place each DNeasy 96 Plate in the correct orientation on a new rack of elution microtubes RS (provided), add 100 µl Buffer AE to each sample, and seal the DNeasy 96 Plates with new AirPore Tape Sheets (provided). Incubate for 1 min at room temperature (15-25℃). Centrifuge for 2 min at 6000 rpm （除去胶布。稀释DNA ，将每个DNeasy 96 Plate 按照正确方向放置在新的稀释微管RS 上，在每个样品中加入100 µl 缓冲液AE ，用新的AirPore Tape Sheets 将DNeasy 96 Plates 密封，在15-25℃ 室温下温育1 min 。在6000 rpm 下离心2 min ）。

Elution in 2×50 µl (instead of 2×100 µl) increases DNA concentration, but decreases the overall DNA yield（稀释在2×50 µl而非2×100 µl可提高DNA 浓度，但在总体上会降低DNA 产量）。

19 、Repeat step 18 with a further 100µl Buffer AE （用100 µl 缓冲液AE 进一步重复第18 步）。

Use new caps (provided) to seal the elution microtubes RS for storage（用新盖子密封稀微型管RS进行贮存）。

DNA storage conditions （DNA 贮存条件）。

DNA is stable for several days when stored at 4℃ in Buffer AE. For long term storage, freezing at -20℃ is recommended（DNA 在4℃缓冲液AE可稳定贮存数天。长期贮存建议在-20℃进行）。