RealPure植物(wù)基因組DNA提取試劑盒
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貨号
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名稱
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規格
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RTG2404-01
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RealPure植物(wù)基因組DNA提取試劑盒
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50次
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● 試劑盒内容：
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試劑盒組成
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RTG2404-01（50次）
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緩沖液AP1
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25 ml
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緩沖液AP2
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10 ml
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緩沖液AP3(濃縮液)
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15 ml
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漂洗液PW（濃縮液）
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25 ml
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洗脫緩沖液EB
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15 ml
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RNase A（10
mg/ml）
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0.5 ml
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吸附柱CG（白(bái)色）
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50個(gè)
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過濾柱CF
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50個(gè)
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收集管
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100個(gè)
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說明書
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1份
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● 儲存條件(jiàn)和效期：
本試劑盒在常溫（25℃左右）幹燥條件(jiàn)下(xià)，可保存1年(nián)；更長(cháng)時間的保存可置于2–8℃。2–8℃保存條件(jiàn)下(xià)，若溶液産生(shēng)沉澱，應在使用前置于37℃下(xià)溶解沉澱至溶液澄清後再使用。
單獨包裝的RNaseA收到(dào)後-20℃保存。
● 産品簡介：
本試劑盒采用可以特異性結合DNA的離心吸附柱和獨特的緩沖液系統，能(néng)提取多(duō)種植物(wù)細胞/組織中的基因組DNA。離心吸附柱可以高(gāo)效、專一(yī)吸附DNA，可最大限度去除雜(zá)質蛋白(bái)及細胞中其他有機(jī)化合物(wù)。提取的基因組DNA片段大，純度高(gāo)，質量穩定可靠。
使用本試劑盒回收的DNA可适用于各種常規操作，包括酶切、PCR、文庫構建、Southern雜(zá)交等實驗。
● 提取得率：
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材料
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DNA得量（μg/100mg）
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Arabidopsis
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3–4 μg
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8–12 μg
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Maize
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15-20
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Pine
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20-25
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Potato
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4-6
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Rape
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2-4
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Spinach
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5-10
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Tobacco
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20-25
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Wheat
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25-30
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● 準備工(gōng)作：
1. 準備液氮；65℃水(shuǐ)浴；無水(shuǐ)乙醇；1.5ml滅菌離心管。
2. 按照(zhào)标簽所示在緩沖液AP3和漂洗液PW中加入無水(shuǐ)乙醇，混勻後蓋緊瓶蓋後常溫貯存備用。
3. 每次使用前請檢查緩沖液AP1，緩沖液AP2和緩沖液AP3是否有沉澱生(shēng)成，如果出現沉澱，37℃溫浴至沉澱溶解後再使用。
● 操作步驟：
如非指出，所有離心步驟均為(wèi)使用台式離心機(jī)在常溫下(xià)離心。
1. 取适量植物(wù)新鮮組織0.5-1g加入液氮充分研磨，取約100mg粉末置于1.5ml離心管中，加入400 μl 緩沖液AP1和6
μl RNaseA (10mg/ml)，漩渦震蕩1分鍾。
2. 将離心管放(fàng)在65℃水(shuǐ)浴10分鍾，水(shuǐ)浴過程中颠倒離心管以混合樣品數次。
3. 加入130
μl 緩沖液AP2，颠倒混勻，冰浴5分鍾。
4. 12,000rpm離心5分鍾。
注：此步驟離心去除去垢劑，蛋白(bái)以及多(duō)糖等雜(zá)質。
5. 取上(shàng)清（通(tōng)常為(wèi)400
μl）轉移到(dào)過濾柱CF中，12,000rpm
離心1分鍾。
6. 将濾出液轉移到(dào)1.5
ml離心管中，加入1.5倍體積（例如400
μl的上(shàng)清液加600 μl緩沖液AP3）緩沖液AP3（使用前請注意是否已經加入無水(shuǐ)乙醇），立即颠倒混勻，簡短離心以去除管蓋内壁的水(shuǐ)珠。
7．吸取步驟6中的混合液加入到(dào)吸附柱CG中（吸附柱放(fàng)入收集管中），12,000
rpm離心1分鍾，倒掉廢液，吸附柱CG放(fàng)回收集管中。
注：離心吸附柱的最大容積是700μl，如果一(yī)次不能(néng)完全上(shàng)柱，可以分次上(shàng)柱離心，保證全部溶液全部加到(dào)離心柱中。
8．向吸附柱CG中加入500
μl漂洗液PW（使用前請先檢查是否已加入無水(shuǐ)乙醇），12,000 rpm離心1分鍾，倒掉廢液，吸附柱CG放(fàng)入收集管中。
9．重複步驟8。
10. 可選步驟：如果離心柱的吸附膜上(shàng)有顔色，向吸附柱CG中加入500
μl無水(shuǐ)乙醇，12,000 rpm離心1分鍾，倒掉廢液，吸附柱CG放(fàng)入收集管中。
11．将吸附柱CG放(fàng)回廢液收集管中，12,000 rpm離心2分鍾。
注：此步驟非常重要，目的是将吸附柱中殘餘的漂洗液去除。漂洗液中乙醇的殘留會(huì)影響後續的酶反應（酶切、PCR等）實驗。
12．将吸附柱CG轉入一(yī)個(gè)幹淨的1.5ml離心管中，向吸附膜的中間部位懸空滴加50-100 μl經65℃水(shuǐ)浴預熱的洗脫緩沖液EB，常溫放(fàng)置2分鍾，12,000
rpm g離心2分鍾。
注；1. 洗脫緩沖液體積最好不少于50 μl，體積過小(xiǎo)影響回收效率。
2. 洗脫液的pH值對于洗脫效率有很大影響。若用水(shuǐ)做洗脫液應保證其pH值在7.0-8.5範圍内，pH值低(dī)于7.0會(huì)降低(dī)洗脫效率。
13. DNA産物(wù)-20℃保存。
● DNA濃度及純度檢測：
基因組DNA片段的大小(xiǎo)與樣品保存時間、操作過程中的剪切力等因素有關。提取的DNA片段可用瓊脂糖凝膠電(diàn)泳和紫外分光(guāng)光(guāng)度計檢測濃度與純度。可配制0.8-1.0％瓊脂糖凝膠，使用λ/HindIII判斷基因組的大小(xiǎo)，完整的基因組大小(xiǎo)應在23kb以上(shàng)。使用分光(guāng)光(guāng)度計檢測時，
OD260/OD280比值應為(wèi)1.7–1.9之間，如果洗脫時不使用洗脫緩沖液，而使用去離子水(shuǐ)洗脫，比值可能(néng)偏低(dī)，但并不表明DNA純度不高(gāo)。
核酸提取産品發表文章
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Product: RTP2201 瓊脂糖凝膠回收試劑盒
Journal: Letters in Applied Microbiology 2009,49,235-240
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Product: RTP2202 PCR産物(wù)純化試劑盒
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Product: RTP2202 PCR産物(wù)純化試劑盒
Journal: Mol Biol Rep (2013) 40:97–107
Institution：Yunnan Institute
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