Name: removal of exogenous materials from the outer portion of frozen cores to investigate the ancient biological communities harbored inside
Uploaded: 2016-07-03T14:00:00.000+00:00
Duration: 9 min 6 s
Description: Watch this Scientific Journal Video about Removal of Exogenous Materials from the Outer Portion of Frozen Cores to Investigate the Ancient Biological Communities Harbored Inside at JoVE.com

This work was funded through the U.S. Army Engineer Research and Development Center, Basic Research Program Office. Permission for publishing this information has been granted by the Chief of Engineers.

1.设备的准备和冻土核心种质<ol><li>设备的准备和现场样品采集和保存减速<ol><li>通过将驱动器适配器插入筒的顶端和转动杠杆锁定到位组装为样品采集螺旋钻。针适配器管到驱动适配器和电机引脚到适配器管。插入桶上的刀具。 </li><li>穿轻型服，丁腈手套，口罩和任何污染，减少对样品。耳罩和在进入隧道冻土（ 图1）安全性的安全帽。 </li><li>进入隧道，并选择收集样本（ 图1B）的位置。 注意:对于垂直或水平钻孔位置，选择已知有证据表明，该材料被冻结（ 例如 ，冰或多年冻土）的区域，也没有已知的大根系统，并且没有已知的砾石。移除所有从生活区的植物材料收集样品之前。如果垂直或水平钻孔中，选择相对平坦，并与螺旋推运器可访问的区域。 </li><li>通过与已用70％的异丙醇，DNA的去污溶液，和RNase去污溶液的塑料材料成层地制备工作站。 </li><li>放置纵向在工作站上半个直径10厘米的聚氯乙烯（PVC）管切割，以提供一个槽，因为它们是从螺旋推运器除去以保持芯。除污PVC管，用70％的异丙醇，DNA净化解决方案，和RNase净化解决方案。 </li><li>附近的工作站，用70％的异丙醇，DNA净化解决方案，并核糖核酸酶净化解决方案喷洒消毒螺旋。从与擦拭螺旋钻移除的解决方案。 </li></ol></li><li>冰和冻土核心的采集和存储<ol><li>选择墙样品的面积（参见1.2.1注意）。 </li><li>用70％的异丙醇，DNA净化解决方案，并核糖核酸酶净化解决方案擦拭消毒冻结壁面积约直径10厘米。 </li><li>提升净化螺旋推运到感兴趣的区域，使得其垂直于样品区，并开始钻入壁（ 图1C，D） ​​的清洁面。 </li><li>小心地从样品采集区域的螺旋。断开电机螺旋并将无菌的PVC管上面的螺旋在洁净工作台。小心倾斜螺旋推运器，使得冷冻芯滑出到无菌的PVC管（ 图1E）。 </li><li>净化用70％的异丙醇，DNA净化解决方案，并核糖核酸酶净化解决方案手套。 </li><li>拿起冰和冻土内核和无菌手套，将它们放入无菌袋。 </li><li>放置在核心凉爽或寒冷的房间里，直到它们被运或处理。 </li><li>嘘的ip使用干冰以保持它们在低于0℃的温度下冷冻芯。 </li><li>紧随核储存在-80℃。 </li></ol></li></ol> 2，多年冻土和冰芯处理<ol><li>备料<ol><li>通过在450℃下进行4小时的烘箱中烘烤制备无菌的，核酸游离重负荷铝箔，金属架，玻璃器皿，金属镊子，和玻璃棉。直至使用抛开这些材料。 </li><li>通过0.22微米过滤器传递溶液消毒95％乙醇和超纯水。 </li><li>在-20℃和水商店乙醇溶液在4℃下。 </li><li>通过用70％乙醇，DNA的去污溶液，和RNase去污溶液喷洒和各溶液后，立即擦拭消毒一个塑料尺。 </li></ol></li><li>细菌培养制备<ol><li>准备肉汤和板成长粘质沙雷氏菌 。 <ol><li>对于肉汤，结合5克试ptone，1克葡萄糖，5克酵母提取物和1g磷酸氢二钾，然后加蒸馏水至达到1L。为了使板，琼脂15克的加入到上述混合物。对于肉汤和板材，调节pH至7和高压釜在121℃下15分钟。 </li></ol></li><li>通过组合8克氯化钠，0.2克氯化钾，1.44加入磷酸氢二钠的，及0.24g的磷酸二氢钾制备1X磷酸盐磨光盐溶液（PBS）中，并加蒸馏水至达到800毫升调节pH至7.4，并添加蒸馏水于121℃以达到1升高压灭菌15分钟。 </li><li>通过浸入S.接种用无菌环肉汤沙雷氏菌培养以前存储在-80℃下冷冻。在无菌条件下，串行稀加入1 ml培养至9毫升的PBS并手动培养反转溶液。 1ml该稀释液加入到9毫升的PBS并手动反转溶液。重复八点多，直到10 -9 DILU达到化。 </li><li>通过1毫升肉汤散发到板，并用细胞吊具扩频传播10 -6，10 -7，10 -8和10 -9解到琼脂平板上。每稀释三次做到这一点。在4℃保存原有的文化。 </li><li>在30℃孵育板24小时。计数菌落数来计算在原始培养细胞的数目。注意:菌落在原始培养的数量将依赖于细菌的生长速度。 </li><li>吸取250微升原生态文化的进入无菌1.5 ml离心管。通过添加1×PBS中的足够量如通过菌落计数在先前步骤中确定的，以获得约10 9个细胞/ ml稀释原始培养。通过以2,500 xg离心离心10分钟沉淀在离心管的细胞。 注:1×PBS中的量将依赖于细菌的生长速度各不相同。 </li><li>在无菌biohood，吸取了肉汤和悬浮细胞在1毫升1×PBS缓冲液。储存在4℃直至使用或约2个月。 </li><li>上处理的当天，淡化S.通过加入1ml的原始S的从步骤2.2.7 沙雷氏菌培养沙雷氏菌培养至39毫升1×PBS缓冲液在50毫升离心管中。 </li></ol></li><li>准备冷室空间，核心处理和保存齿轮<ol><li>前冷却的冷室，洁净的墙壁，地板和金属表用1％漂白溶液中。 </li><li>冷室的设定温度至约-11℃。 </li><li>一旦冷室已达到所需温度时，穿轻型服，腈手套和口罩，以任何污染减少到冷室和样品。 注:两个身着正常个人需要这种方法。 </li><li>把无菌材料 （ 如铝箔，金属架，玻璃器皿，盘， 粘质沙雷氏菌文化，MIcrotome刀片）和样品入冷室，地点在无菌表。 </li></ol></li><li>多年冻土和冰核处理在寒冷的房间设施<ol><li>放置一个永久冻土芯上的无菌的，核酸自由铝箔的片材。 </li><li>轻轻接种芯的外侧与S的稀培养沙雷氏菌使用无菌泡沫塞（ 图2B）。 </li><li>放在一个盘上方坐在无菌金属机架的核心。 </li><li>消毒用70％乙醇，DNA的去污溶液，和RNase去污溶液钢切片机刀片。 </li><li>有70％的乙醇，DNA净化解决方案，并核糖核酸酶净化解决方案个体干净丁腈手套，并在45°角的盘上方持有的核心。 </li><li>有个人乙轻轻刮去约5mm的整个芯的外侧的，包括端部，使用无菌刀片而个人A熄灭各刮后的核心（ <str翁&gt;图2C和图3A，B）。根据需要擦拭用70％的乙醇，DNA的去污溶液，和RNase去污溶液刀片。 </li><li>有个人乙小心，并迅速倒入过滤除菌的95％的乙醇对核心，而个人A熄灭的溶液倾倒芯（ 图2D，3C）。 </li><li>有个别乙立即冲洗用过滤灭菌的水的核心。 </li><li>用新的无菌金属架更换托盘使用的金属架。 </li><li>有个别A以45°角的托盘上方清理他或她的丁腈手套用70％乙醇，DNA净化解决方案，并核糖核酸酶净化解决方案和保持核心。 </li><li>有个别乙喷洒DNA净化解决方案的整个核心。 </li><li>有个别乙立即冲洗用过滤灭菌的水的核心。 </li><li>有个别乙喷洒核糖核酸酶净化解决方案的整个核心。 </li><li>有Individu人乙立即冲洗用过滤灭菌的水的核心。 </li><li>放置在铝箔的无菌片芯和轻轻包。 </li></ol></li><li>解冻外核<ol><li>将在无菌玻璃皿无菌金属架在层流无菌biohood。 </li><li>将具体到S. 2琼脂平板沙雷氏菌在无菌机架下面的玻璃皿从核心（ 图2E）收集液体。 </li><li>放置在无菌的金属架的核心。 </li><li>允许约2-5毫米芯的外表面，以在23℃解冻（平均，这将约10分钟内发生）。转芯大约每2分钟90°。 </li><li>使用无菌镊子和无菌玻璃棉拭芯的整个表面上，并接种2琼脂平板特定至S.由这些材料擦拭到板的表面沙雷氏菌 。接种与原来的文化两个新的板块用于掺杂的外芯，将其暴露于冷室的低温条件下。 </li><li>通过将无菌标尺附近，但不触及核心测量解冻圆柱形芯的外形尺寸。 </li><li>将核心成大无菌袋，并将其存储在-80℃。 </li></ol></li><li>检查增长<ol><li>在23℃孵育琼脂平板一周。 </li><li>检查S的生长琼脂平板沙雷菌菌落。 <ol><li>如果有在平板上没有可见的菌落，继续执行步骤2.5.3。 </li><li>如果菌落出现在平板，从步骤2.1.1第2条"冻土和冰芯处理"重复。 </li></ol></li><li>获得从冷冻芯，并无菌转移到一个无菌袋。 </li><li>存储在无菌袋中的芯在4℃下约24-48小时，解冻整个核心。 </li></ol></li></ol> 3.获取子样本从冰芯和冻土核酸提取<ol><li>从冰芯核酸提取<ol><li>轻轻摇动袋（ 图2G）混合从冰芯解冻材料。 </li><li>倒入解冻材料放入无菌容器用0.22微米的过滤器在真空下收集的微生物。 </li><li>无菌取出用无菌镊子过滤，并将其放置在无菌2毫升陶瓷珠管（1.4毫米）。 </li><li>储存在-80℃的过滤器。 </li></ol></li><li>冻土核核酸提取<ol><li>通过轻轻揉捏袋子从混合冻土核心解冻材料。 </li><li>多年冻土已充分混合后，得到大约为0.5 g整体土壤的子样本用无菌scoopula并将其放置在该直立坐在一个平衡配衡无菌2毫升陶瓷珠螺旋盖管（1.4毫米）。 </li><li>店子样本在-80℃。 </li><li>获取样本的重量含水量。 <ol><li>措施10克永久冻土带与不孕在天平上去皮重的锡Ëscoopula和地点。测量永久冻土和锡的湿重。 </li><li>在24小时设定在105℃的烘箱孵育永久冻土。测量永久冻土和锡干质量。 </li><li>由永久冻土层的干质量减去冻土的湿重和多年冻土的干燥质量除以计算重量含水量。 </li></ol></li><li>在-80°C储存永久冻土带。 </li></ol></li></ol> 4.永久冻土和冰芯中提取核酸<ol><li>材料准备<ol><li>制备琥珀小瓶通过用0.1％焦碳酸二乙酯（DEPC）处理，在37℃，高压灭菌孵育O / N保存的解决方案。 </li><li>制备240 mM磷酸钾缓冲液。添加2.5克氢磷酸二氢和38.7克磷酸氢二钾的。通过加入无菌水调节终体积为500毫升。 </li><li>准备溴化十六（CTAB）提取BUFFER 4.1 5g氯化钠溶解在80毫升水中，慢慢加入10克CTAB同时加热和搅拌。通过加入无菌水调节最终体积至100ml。 </li><li>添加的磷酸缓冲液等体积​​的CTAB缓冲液和过滤用0.22微米的过滤器消毒。盖瓶用铝箔和储存在室温。 </li><li>通过组合9.35克氯化钠和100ml无菌水制备1.6M的氯化钠（NaCl）溶液。加入10克聚乙二醇8000至1.6M的NaCl溶液和过滤消毒。保存在4℃。 </li></ol></li><li>根据Griffiths 等人的修改的版本的核酸提取22和TOWE 等人 23 <ol><li>穿轻型服，丁腈手套，和口罩。干净的实验室空间并用70％的乙醇，DNA的去污溶液，和RNase去污溶液移液管。 </li><li>在2毫升删除子样本（来自冰芯或永久冻结带的土壤样品过滤器），陶瓷珠螺-CA从-80℃冷冻室和解冻在RT p管。 </li><li>将0.5ml溴化十六（CTAB）提取液，涡旋简要的。 </li><li>将0.5ml酚 - 氯仿 - 异戊醇（25:24:1）（pH 8）中，并使用上的涡旋平板适配器水平摇晃试管10分钟。 </li><li>在4℃以下珠跳动，离心管在16100 XG 5分钟。 </li><li>到一个新的无菌1.5毫升管除去水层，并用等体积的氯仿 - 异戊醇混合（24:1）。离心16100 XG在4℃下5分钟。 </li><li>到一个新的无菌1.5毫升管除去水层，加入30％的聚乙二醇8000和1.6 M氯化钠的两卷。孵育在4℃下2小时。 </li><li>离心机在16100 xg离心在4℃下10分钟。 </li><li>于4℃在16100 xg离心用约500微升冰冷的70％乙醇和离心机的洗涤核酸沉淀10分钟。 </li><li>在2小时的无菌biohood空气干燥颗粒。重悬在50μlDNA酶/无RNase的TE缓冲液（pH8.0）中。 DNA可用于下游应用如PCR和qPCR。 </li><li>确定的DNA的浓度用分光光度计。 </li><li> DNA稀释用无菌，不含DNA的水达到每20反应纳克。 </li></ol></li></ol>

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The authors have nothing to disclose.

冰雪圈提供了保存完好的生物，过去的环境条件下坚持访问。虽然恢复类群可能不是完整的历史社会，来自冰川和冻土样品的分析中回收可产生约选择时间段15-16宝贵的历史资料。举例来说，有意义的生物信息已经从冰研究在格陵兰冰盖考察20厌氧活性和冻土试验研究了碳循环过程的解冻33的结果绘制的，并已在白令陆桥16提供了深入了解真菌的多样性冻土。之前进...

冰雪圈（ 例如 ，永冻层土壤，冰功能，冰川积雪，积雪和冰）提供了一个窥探到过去的环境条件下坚持什么类型的生物。由于这些基材可以是几十到几十万岁，他们的微生物群落，因为沉积在冷冻保存，反映古环境条件。要正确分析这些生态系统和提取冷冻样品的冻土和冰块，正确的收集和处理有意义的生物信息是必要的。这是最重要的，因为气候预测为21 世纪指示北极和亚北极地区1有明显的变暖的潜力。具体来说，内政部阿拉斯加和格陵兰预期升温5℃左右，并分别在2100年2,3 7℃。预计这将影响显著土壤和水生微生物群落，因此，相关的生物地球化学过程。温暖的温度和降水改变政权有望开始在许多领域2-5冻土退化可能导致较厚，季节性解冻（活动）6,7层，冻土的解冻，和大量的冰体，如熔化地下冰，冰楔，和隔离冰8。这将极大地改变除了这些生态系统中的植物和动物的生物多样性生物地球化学属性。 冰川和永冻层同生沉积物和冰的功能已经被困化学和代表的环境中生活是什么在那里形成的功能时的生物学证据。例如，在内部阿拉斯加，既Illinoisan和威斯康星州的永久冻土岁都存在这个永久冻土尤其在本（YBP）包含IMPA的生物地球化学的证据之前，提供了从现代到15万年约会不同的位置对生物多样性过去气候变化克拉。其结果是，这些沉积物提供生物地球化学和生物多样性的记录了数千年。由于该区域具有低的沉积速率，并从来没有被冰川覆盖的，不受干扰的样本是收集和分析，无论是钻访问垂直插入隧道土壤剖面或钻孔水平。更重要的是，大量的记录存在的突出特别是在该地区永久冻土9-14的独特生物地球化学特征。具体来说，DNA分析的应用，同时估计现存的古冰和永冻土样本中的生物多样性的存在和程度使古环境条件特定生物联动和栖息地的职业探索。 以前的研究已经确定在哺乳动物，植物和可追溯至50K YBP 11，15-19样品微生物气候影响，虽然每个研究使用各色一nt的方法来收集和净化冻土和冰芯。在某些情况下，钻芯灭菌16，20-21，虽然具体的方法没有澄清是否外来核酸还从样品中消除。在其他研究中，细菌分离物15（ 例如 ， 粘质沙雷氏菌 ），以及22已被用于测量的净化程序的功效荧光微球。 这个实验是一个大型研究调查冻土样本可以追溯到大约40K YBP微生物群落的组成部分。研究这部分的具体目标是成功地净化冰和冻土内核。就我们所知，没有任何方法集成了使用而设计的，以消除从冷冻芯的外侧部分外来核酸和相关的核酸溶液。尽管这是一个事实，这些解决方案是commonlŸ用于净化实验室设备分子实验。 一旦芯进行了去污，使用由Griffiths等 23和TOWE 等人 24开发的协议提取基因组DNA，使用分光光度计进行定量，并用无菌，无DNA的水稀释至达到每反应20纳克。细菌的16S rRNA基因用引物331F及797R扩增和探针BacTaq 25和古16S rRNA基因用引物拱349F和拱806R和在下列条件下探测TM拱516F 26扩增:95℃600秒，随后45个循环95℃，30秒，57℃60秒，72℃，在40℃，30秒下进行25秒，最后延伸。所有的qPCR反应中重复进行。在20微升的反应体积包含20 ng的DNA，引物10μM的，探针的5微米，和10微升的qPCR反应混合物。 FO标准- [R采用基因组DNA分别假单胞菌荧光和嗜盐salinarum，细菌和古细菌定量PCR制备。两者中生长至对数期。平板计数进行了与DNA从培养物中分离。与一个的假设和每个基因组为H的16S rRNA基因的六份分光光度计基因组DNA进行定量salinarum和P.荧光 ，分别为27-28。细菌和古细菌的基因的拷贝数是基于标准曲线上计算出的，对数转换，以占治疗之间不等方差，并通过ANOVA评估。 群落的组合物通过使用流动池和桥扩增技术测序16S rRNA基因，并与"定量分析上市微生物生态学&#39;（QIIME）29分析该社区确定。正向和反向读取被连接在一起，然后序列进行过滤，索引，并选择高品质的代表从头操作分类单元（OTU）通过序列比对分配有一个参考数据库。比对的序列进行了比较，对分类分配一个单独的参考数据库。一个门级OTU表的建立是为了确定一般群落组成。

<table><tbody><tr><td>Auger</td><td>Snow, Ice, and Permafrost Research Establishment (SIPRE), Fairbanks, AK</td><td></td><td></td></tr><tr><td>70% Isopropanol</td><td>Walmart</td><td>551116880</td><td></td></tr><tr><td>95% Ethyl Alcohol (denatured)&amp;nbsp;</td><td>Fisher Scientific, Pittsburgh, PA</td><td>A407-4</td><td></td></tr><tr><td>DNA decontamination solution, DNA Away</td><td>Molecular Bio-Products, Inc., San Diego, CA</td><td>7010</td><td></td></tr><tr><td>RNase decontamination solution, RNase Away</td><td>Molecular Bio-Products, Inc., San Diego, CA&amp;nbsp;</td><td>7002</td><td></td></tr><tr><td>Light Duty Suits</td><td>Kimberly-Clark Professional, Roswell, GA</td><td>10606</td><td></td></tr><tr><td>Nitrile Gloves</td><td>Fisher Scientific, Pittsburgh, PA</td><td>FFS-700</td><td></td></tr><tr><td>Antiviral Masks</td><td>Curad, Walgreens</td><td>CUR3845</td><td></td></tr><tr><td>Sterile Sample Bags&amp;nbsp;</td><td>Nasco, Fort Atkinson, WI</td><td>B01445</td><td></td></tr><tr><td>Steel Microtome Blade&amp;nbsp;</td><td>B-Sharp Microknife, Wake Forest, NC</td><td></td><td></td></tr><tr><td>Metal Rack</td><td>Fabricated at CRREL, Hanover, NH</td><td></td><td></td></tr><tr><td>Tray</td><td>Handy Paint Products, Chanhassen, MN</td><td>7500-CC</td><td></td></tr><tr><td>Aluminum Foil</td><td>Western Plastics, Temecula, CA</td><td></td><td></td></tr><tr><td>500 ml Bottle with 0.22&amp;nbsp;&amp;mu;m Filter</td><td>Corning, Corning, NY</td><td>430513</td><td></td></tr><tr><td>&lt;em&gt;Serratia marcescens&lt;/em&gt;</td><td>ATCC, Manassas, VA</td><td>17991</td><td></td></tr><tr><td>Biosafety Hood</td><td>NuAire, Plymouth, MN</td><td>NU-425-400</td><td></td></tr><tr><td>Petri Dish</td><td>Fisher Scientific, Pittsburgh, PA</td><td>FB0875712</td><td></td></tr><tr><td>ATCC Agar 181- Tryptone</td><td>Acros Organics, NJ</td><td>61184-5000</td><td></td></tr><tr><td>ATCC Agar 181- Glucose</td><td>Fisher Scientific, Pittsburgh, PA</td><td>BP381-500</td><td></td></tr><tr><td>ATCC Agar 181- Yeast Extract</td><td>Fisher Scientific, Pittsburgh, PA</td><td>BP1422-500</td><td></td></tr><tr><td>ATCC Agar 181- Dipotassium Phosphate</td><td>JT Baker, Phillipsburg, NJ</td><td>3252-01</td><td></td></tr><tr><td>ATCC Agar 181- Agar</td><td>Difco, Sparks, MD</td><td>214530</td><td></td></tr><tr><td>NanoDrop 2000 UV Vis Spectrophotometer</td><td>Thermo Fisher Scientific, Wilmington, DE</td><td></td><td></td></tr><tr><td>Lightcycler 480 System</td><td>Roche Molecular Systems, Inc., Indianapolis, IN</td><td></td><td></td></tr><tr><td>&lt;em&gt;Halobacterium salinarum&lt;/em&gt;</td><td>American Type Culture Collection (ATCC), Manassas, VA</td><td></td><td></td></tr><tr><td>&lt;em&gt;Pseudomonas fluorescens&lt;/em&gt;</td><td>American Type Culture Collection (ATCC), Manassas, VA</td><td></td><td></td></tr><tr><td>Microbial DNA Isolation Kit</td><td>MoBio Laboratories, Carlsbad, CA</td><td>12224-50</td><td></td></tr><tr><td>Ear Protection</td><td>Elvex</td><td>EP-201</td><td></td></tr><tr><td>Hard Hat</td><td></td><td></td><td></td></tr><tr><td>Kimwipes</td><td>Kimberly-Clark Professional, Roswell, GA</td><td>34705</td><td></td></tr><tr><td>Glass Wool</td><td>Pyrex</td><td>430330</td><td></td></tr><tr><td>Ruler</td><td></td><td></td><td></td></tr><tr><td>Weighing Tin&amp;nbsp;</td><td>Fisher Scientific, Pittsburgh, PA</td><td>08-732-100</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma Aldrich, St Louis, MO</td><td>S-9625</td><td></td></tr><tr><td>Potassium chloride</td><td>JT Baker, Phillipsburg, NJ</td><td>3040-04</td><td></td></tr><tr><td>Potassium phosphate, monobasic</td><td>JT Baker, Phillipsburg, NJ</td><td>&amp;nbsp;3246-01</td><td></td></tr><tr><td>Potassium phosphate, dibasic</td><td>JT Baker, Phillipsburg, NJ</td><td>3252-01</td><td></td></tr><tr><td>Sodium phosphate dibasic, anhydrous</td><td>Fisher Scientific, Pittsburgh, PA</td><td>BP332-500</td><td></td></tr><tr><td>50 ml Centrifuge Tubes</td><td>Corning, Corning, NY</td><td>4558</td><td></td></tr><tr><td>2 ml Microcentrifuge Tubes</td><td>MoBio Laboratories, Carlsbad, CA</td><td>1200-250-T</td><td></td></tr><tr><td>2 ml Ceramic Bead Tubes (1.4&amp;nbsp;mm)</td><td>MoBio Laboratories, Carlsbad, CA</td><td>13113-50</td><td></td></tr><tr><td>Scoopula</td><td>Thermo Fisher Scientific, Wilmington, DE</td><td>1437520</td><td></td></tr><tr><td>Balance</td><td>Ohaus, Parsippany, NJ</td><td>E12130</td><td></td></tr><tr><td>Diethylpyrocarbonate (DEPC)</td><td>Sigma Aldrich, St Louis, MO</td><td>D5758</td><td></td></tr><tr><td>Hexadecyltrimethylammoniabromide (CTAB)&amp;nbsp;</td><td>Acros Organics, NJ</td><td>22716-5000</td><td></td></tr><tr><td>Polyethylene glycol 8000&amp;nbsp;</td><td>Sigma Aldrich, St Louis, MO</td><td>P5413-1KG</td><td></td></tr><tr><td>Phenol-chloroform-isoamyl alcohol (25:24:1) (pH 8)&amp;nbsp;</td><td>Fisher Scientific, Pittsburgh, PA</td><td>BP1752-400</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf, Hauppauge, NY</td><td>5417R</td><td></td></tr><tr><td>Chloroform-isoamyl alcohol (24:1)</td><td>Sigma Aldrich, St Louis, MO</td><td>C0549-1PT</td><td></td></tr><tr><td>TE Buffer</td><td>Ambion (Thermo Fisher), Wilmington, DE</td><td>AM9860</td><td></td></tr><tr><td>Pipets</td><td>Rainin, Woburn, MA</td><td>Pipet Lite XLS, 2, 10, 200, and 1,000 &amp;micro;l pipets</td><td></td></tr><tr><td>Pipet tips</td><td>Rainin, Woburn, MA</td><td>Rainin LTS presterilized, low retention, filtered tips, 10, 20, 200, 1,000&amp;nbsp;&amp;micro;l</td><td></td></tr><tr><td>Vortexor</td><td>Scientific Industries, Bohemia, NY</td><td>G-560</td><td></td></tr><tr><td>Vortex Adaptor</td><td>MoBio Laboratories, Carlsbad, CA</td><td>13000-V1</td><td></td></tr><tr><td>Clear Bottle</td><td>Corning, Corning, NY</td><td>C1395500</td><td></td></tr><tr><td>Amber Bottle</td><td>Corning, Corning, NY</td><td>C5135250</td><td></td></tr><tr><td>Bottle Top Filters, 0.22&amp;nbsp;&amp;micro;m</td><td>Corning, Corning, NY</td><td>430513</td><td></td></tr><tr><td>60 ml Syringe</td><td>Becton, Dickenson and Company, Franklin Lakes, NJ</td><td>BD 309653</td><td></td></tr><tr><td>Millex Syringe filters, 0.22 &amp;mu;m</td><td>EMD Millipore, Billerica, MA</td><td>SLGV033RB</td><td></td></tr><tr><td>70% Ethanol</td><td>Fisher Scientific, Pittsburgh, PA</td><td>BP2818-500</td><td>diluted &amp;amp; filter sterilized</td></tr><tr><td>Isotemp 100 L Oven</td><td>Fisher Scientific, Pittsburgh, PA</td><td>151030511</td><td></td></tr><tr><td>Cell Spreader</td><td>Fisher Scientific, Pittsburgh, PA</td><td>08-100-10</td><td></td></tr><tr><td>Disposable Inoculating Loops</td><td>Fisher Scientific, Pittsburgh, PA</td><td>22-363-602</td><td></td></tr></tbody></table>

removal of exogenous materials from the outer portion of frozen cores to investigate the ancient biological communities harbored inside

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect conditions over vast time periods and investigation of biological materials harbored inside could provide insight of ancient environments. To appropriately analyze these ecosystems and extract meaningful biological information from frozen soils and ice, proper collection and processing of the frozen samples is necessary. This is especially critical for microbial and DNA analyses since the communities present may be so uniquely different from modern ones. Here, a protocol is presented to successfully collect and decontaminate frozen cores. Both the absence of the colonies used to dope the outer surface and exogenous DNA suggest that we successfully decontaminated the frozen cores and that the microorganisms detected were from the material, rather than contamination from drilling or processing the cores.

该方法可用于净化各种冰冻圈环境中收集的冷冻样品，从冰川冻土到。在这里，我们提出从工程技术研究开发中心收集冰和永冻土样本专门收集的数据-寒冷地区研究和工程实验室（ERDC-CRREL）冻土隧道位于福克斯，AK（ 图1A和1B）。多年冻土隧道约110米延伸到黄金溪山谷的一侧，并提供访问丰富的冰和淤泥冲积层30-31。从冰楔和冰冻土壤样?...

Watch this Scientific Journal Video about Removal of Exogenous Materials from the Outer Portion of Frozen Cores to Investigate the Ancient Biological Communities Harbored Inside at JoVE.com

从冷冻核心外部去除外源物质以调查内部的古代生物群落生物学 |视频

Removal of Exogenous Materials from the Outer Portion of Frozen Cores to Investigate the Ancient Biological Communities Harbored Inside

冰雪圈提供了保存完好的生物，过去的环境条件下坚持访问。的协议，提出收集和净化土壤和冰的永冻层的核心。缺乏外源菌落和DNA的建议检测微生物所代表的材料，而不是从钻孔或加工污染。

从冷冻芯的外层部分外源物质的去除探讨古代生物群落里面窝藏

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect ...

removal-exogenous-materials-from-outer-portion-frozen-cores-to

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7.9K 次观看.  US Army Engineer Research & Development Center, Hanover, NH. 冰雪圈提供了保存完好的生物，过去的环境条件下坚持访问。的协议，提出收集和净化土壤和冰的永冻层的核心。缺乏外源菌落和DNA的建议检测微生物所代表的材料，而不是从钻孔或加工污染。 

视频: Removal of Exogenous Materials from the Outer Portion of Frozen Cores to Investigate the Ancient Biological Communities Harbored Inside

Sediment Core Sectioning and Extraction of Pore Waters under Anoxic Conditions

We demonstrate a method for sectioning sediment cores and extracting pore waters while maintaining oxygen-free conditions. A simple, inexpensive system is built and can be transported to a temporary work space close to field sampling site(s) to facilitate rapid analysis. Cores are extruded into a portable glove bag, where they are sectioned and each 1-3 cm thick section (depending on core diameter) is sealed into 50 ml centrifuge tubes. Pore waters are separated with centrifugation outside of the glove bag and then returned to the glove bag for separation from the sediment. These extracted pore water samples can be analyzed immediately. Immediate analyses of redox sensitive species, such as sulfide, iron speciation, and arsenic speciation indicate that oxidation of pore waters is minimal; some samples show approximately 100% of the reduced species, e.g. 100% Fe(II) with no detectable Fe(III). Both sediment and pore water samples can be preserved to maintain chemical species for further analysis upon return to the laboratory.

A protocol for sectioning sediment cores and extracting pore waters under anoxic conditions in order to permit analysis of redox sensitive species in both solids and fluids is presented.

沉积物核心切片和孔隙水萃取缺氧条件下

We demonstrate a method for sectioning sediment cores and extracting pore waters while maintaining oxygen-free conditions. A simple, inexpensive system is ...

科研

环境科学

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

从土壤、根际和根 Endosphere 提取细菌群落数据的根菌群探析

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

遗传学

Isolation and Analysis of Microbial Communities in Soil, Rhizosphere, and Roots in Perennial Grass Experiments

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The purpose of this manuscript is to provide detail on how to rapidly sample soil, rhizosphere, and endosphere of replicated field trials and analyze changes that may occur in the microbial communities due to sample type, treatment, and plant genotype. The experiment used to demonstrate these methods consists of replicated field plots containing two, pure, warm-season grasses (Panicum virgatum and Andropogon gerardii) and a low-diversity grass mixture (A. gerardii, Sorghastrum nutans, and Bouteloua curtipendula). Briefly, plants are excavated, a variety of roots are cut and placed in phosphate buffer, and then shaken to collect the rhizosphere. Roots are brought to the laboratory on ice and surface sterilized with bleach and ethanol (EtOH). The rhizosphere is filtered and concentrated by centrifugation. Excavated soil from around the root ball is placed into plastic bags and brought to the lab where a small amount of soil is taken for DNA extractions. DNA is extracted from roots, soil, and rhizosphere and then amplified with primers for the V4 region of the 16S rRNA gene. Amplicons are sequenced, then analyzed with open access bioinformatics tools. These methods allow researchers to test how the microbial community diversity and composition varies due to sample type, treatment, and plant genotype. Using these methods along with statistical models, the representative results demonstrate there are significant differences in microbial communities of roots, rhizosphere, and soil. Methods presented here provide a complete set of steps for how to collect field samples, isolate, extract, quantify, amplify, and sequence DNA, and analyze microbial community diversity and composition in replicated field trials.

Excavation of plant roots from the field as well as processing of samples into endosphere, rhizosphere, and soil are described in detail, including DNA extraction and data analysis methods. This paper is designed to enable other laboratories to use these techniques for the study of soil, endosphere, and rhizosphere microbiomes.

多年生草试验中土壤、根际和根系微生物群落的分离与分析

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The ...

Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to characterize. However, to understand the thermodynamic and kinetic controls on greenhouse gas (carbon dioxide [CO2] and methane [CH4]) production resulting from the decomposition of NOM, a molecular-level characterization coupled with microbial proteome analyses is necessary. Further, climate and environmental changes are expected to perturb natural ecosystems, potentially upsetting complex interactions that influence both the supply of organic matter substrates and the microorganisms performing the transformations. A detailed molecular characterization of the organic matter, microbial proteomics, and the pathways and transformations by which organic matter is decomposed will be necessary to predict the direction and magnitude of the effects of environmental changes. This article describes a methodological throughput for comprehensive metabolite characterization in a single sample by direct injection Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), gas chromatography mass spectrometry (GC-MS), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography mass spectrometry (LC-MS), and proteomics analysis. This approach results in a fully-paired dataset which improves statistical confidence for inferring pathways of organic matter decomposition, the resulting CO2 and CH4 production rates, and their responses to environmental perturbation. Herein we present results of applying this method to NOM samples collected from peatlands; however, the protocol is applicable to any NOM sample (e.g., peat, forested soils, marine sediments, etc.).

This protocol describes a single throughput for complementary analytical and omics techniques culminating in a fully-paired characterization of natural organic matter and microbial proteomics in different ecosystems. This approach permits robust comparisons for identifying metabolic pathways and transformations important for describing greenhouse gas production and predicting responses to environmental change.

用于表征复杂天然有机物混合物的单吞吐量互补高分辨率分析技术

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to ...

A Method to Preserve Wetland Roots and Rhizospheres for Elemental Imaging

Roots extensively interact with their soil environment but visualizing such interactions between roots and the surrounding rhizosphere is challenging. The rhizosphere chemistry of wetland plants is particularly challenging to capture because of steep oxygen gradients from the roots to the bulk soil. Here a protocol is described that effectively preserves root structure and rhizosphere chemistry of wetland plants through slam-freezing and freeze drying. Slam-freezing, where the sample is frozen between copper blocks pre-cooled with liquid nitrogen, minimizes root damage and sample distortion that can occur with flash-freezing while still minimizing chemical speciation changes. While sample distortion is still possible, the ability to obtain multiple samples quickly and with minimal cost increases the potential to obtain satisfactory samples and optimizes imaging time. The data show that this method is successful in preserving reduced arsenic species in rice roots and rhizospheres associated with iron plaques. This method can be adopted for studies of plant-soil relationships in a wide variety of wetland environments that span concentration ranges from trace-element cycling to phytoremediation applications.

We describe a protocol to sample, preserve, and section intact roots and the surrounding rhizosphere soil from wetland environments using rice (Oryza sativa L.) as a model species. Once preserved, the sample can be analyzed using elemental imaging techniques, such as synchrotron X-ray fluorescence (XRF) chemical speciation imaging.

元素成像保护湿地根和岩石圈的方法

Roots extensively interact with their soil environment but visualizing such interactions between roots and the surrounding rhizosphere is challenging. The ...

Optimized Bone Sampling Protocols for the Retrieval of Ancient DNA from Archaeological Remains

The methods presented here seek to maximize the chances for the recovery of human DNA from ancient archaeological remains while limiting input sample material. This was done by targeting anatomical sampling locations previously determined to yield the highest amounts of ancient DNA (aDNA) in a comparative analysis of DNA recovery across the skeleton. Prior research has suggested that these protocols maximize the chances for the successful recovery of ancient human and pathogen DNA from archaeological remains. DNA yields were previously assessed by Parker et al. 2020 in a broad survey of aDNA preservation across multiple skeletal elements from 11 individuals recovered from the medieval (radiocarbon dated to a period of circa (ca.) 1040-1400 CE, calibrated 2-sigma range) graveyard at Krakauer Berg, an abandoned medieval settlement near Pei&#223;en Germany. These eight sampling spots, which span five skeletal elements (pars petrosa, permanent molars, thoracic vertebra, distal phalanx, and talus) successfully yielded high-quality ancient human DNA, where yields were significantly greater than the overall average across all elements and individuals. Yields were adequate for use in most common downstream population genetic analyses. Our results support the preferential use of these anatomical sampling locations for most studies involving the analyses of ancient human DNA from archaeological remains. Implementation of these methods will help to minimize the destruction of precious archaeological specimens.

The protocol presents a series of best practice protocols for the collection of bone powder from eight recommended anatomical sampling locations (specific locations on a given skeletal element) across five different skeletal elements from medieval individuals (radiocarbon dated to a period of ca. 1040-1400 CE, calibrated 2-sigma range).

优化的骨采样方案，用于从考古遗迹中提取古代DNA

The methods presented here seek to maximize the chances for the recovery of human DNA from ancient archaeological remains while limiting input sample ...

<meta charset="utf8"></head><body>树木生态学研究中田间到实验室树木年轮数字化的协议

A New Workflow for Sampling and Digitizing Increment Cores

Here we present a new workflow from taking increment cores in the field, storing and transporting them to the lab, to digitizing their tree rings for further analyses for subsequent dendroecological analyses. The procedure involves the use of new sample carriers for increment cores. These new G&#228;rtner Schneider Core (GSC) holders are designed using three-dimensional (3D) modeling software and finally printed with a 3D printer. Using these mounts from the beginning in the field, the cores can directly be cut with a core microtome, and their surface can then be digitized without further rearrangement using a new high-resolution image-capturing system. They are thus available for direct analysis. This system allows digitizing tree rings from cores and disks, and also taking images from long micro sections (up to 40 cm) using transmitted light. This feature is of special interest for dendroecological and geomorphic applications to identify the onset of any disturbance in micro sections cut with a core-microtome.

<meta charset="utf8"></head><body>作者聚焦：用于推进树木生态学和木材解剖学研究的创新设备开发

We present a protocol to use 3D-printed mounts to fix increment cores in the field without the necessity of unpacking and gluing them on wooden mounts. The new GSC holder allows placing the cores in a core microtome to cut their surface and directly transfer them to digital image capturing.

用于对增量岩芯进行采样和数字化的新工作流程 

Here we present a new workflow from taking increment cores in the field, storing and transporting them to the lab, to digitizing their tree rings for ...

Collection of Frozen Rodent Brain Regions for Downstream Analyses

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex (mPFC) or nucleus accumbens. The challenge in this work is to dissect the correct area while preserving the microenvironment to be examined. In this paper, we describe a simple, low-cost method using resources readily available in most labs. This method preserves nucleic acid and proteins by keeping the tissue frozen throughout the process. Brains are cut into 0.5&#8211;1.0 mm sections using a brain matrix and arranged on a frozen glass plate. Landmarks within each section are compared to a reference, such as the Allen Mouse Brain Atlas, and regions are dissected using a cold scalpel or biopsy punch. Tissue is then stored at -80 &#176;C until use. Through this process rat and mouse mPFC, nucleus accumbens, dorsal and ventral hippocampus and other regions have been successfully analyzed using qRT-PCR and Western assays. This method is limited to brain regions that can be identified by clear landmarks.

This procedure describes the collection of discrete frozen brain regions to obtain high-quality protein and RNA using inexpensive and commonly available tools.

用于下游分析的冷冻啮齿动物大脑区域集合

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex ...

从冷冻芯的外层部分外源物质的去除探讨古代生物群落里面窝藏

摘要

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此视频中的章节

沉积物核心切片和孔隙水萃取缺氧条件下

从土壤、根际和根 Endosphere 提取细菌群落数据的根菌群探析

多年生草试验中土壤、根际和根系微生物群落的分离与分析

用于表征复杂天然有机物混合物的单吞吐量互补高分辨率分析技术

元素成像保护湿地根和岩石圈的方法

优化的骨采样方案，用于从考古遗迹中提取古代DNA

用于对增量岩芯进行采样和数字化的新工作流程

用于对增量岩芯进行采样和数字化的新工作流程

用于对增量岩芯进行采样和数字化的新工作流程

用于下游分析的冷冻啮齿动物大脑区域集合