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Solarbio-酵母質粒小量提取試劑盒 

產品(pin)簡(jian)介：試(shi)(shi)劑(ji)(ji)盒采(cai)用(yong)(yong)(yong)酶法(fa)破碎酵(jiao)(jiao)母(mu)(mu)細(xi)(xi)胞壁和(he)(he)堿裂解法(fa)裂解酵(jiao)(jiao)母(mu)(mu)細(xi)(xi)胞來提取(qu)(qu)酵(jiao)(jiao)母(mu)(mu)質(zhi)(zhi)(zhi)粒(li)(li) DNA。所(suo)采(cai)用(yong)(yong)(yong)的(de)(de)(de)(de)酵(jiao)(jiao)母(mu)(mu)破壁酶能有(you)(you)(you)效(xiao)地(di)破壞酵(jiao)(jiao)母(mu)(mu)細(xi)(xi)胞壁，提高酵(jiao)(jiao)母(mu)(mu)質(zhi)(zhi)(zhi)粒(li)(li) DNA 的(de)(de)(de)(de)產量(liang)。吸(xi)(xi)附(fu)(fu)(fu)(fu)(fu)柱(zhu)(zhu)(zhu)(zhu)(zhu)中(zhong)(zhong)(zhong)采(cai)用(yong)(yong)(yong)的(de)(de)(de)(de)硅基(ji)質(zhi)(zhi)(zhi)材料(liao)能高效(xiao)、專一(yi)地(di)吸(xi)(xi)附(fu)(fu)(fu)(fu)(fu) DNA，可限度去(qu)除(chu)雜質(zhi)(zhi)(zhi)蛋白(bai)質(zhi)(zhi)(zhi)及細(xi)(xi)胞中(zhong)(zhong)(zhong)其(qi)他(ta)有(you)(you)(you)機化(hua)合物(wu)。使(shi)(shi)用(yong)(yong)(yong)本(ben)試(shi)(shi)劑(ji)(ji)盒提取(qu)(qu)的(de)(de)(de)(de)酵(jiao)(jiao)母(mu)(mu)質(zhi)(zhi)(zhi)粒(li)(li) DNA 可適用(yong)(yong)(yong)于(yu)各種(zhong)常規的(de)(de)(de)(de)分(fen)子生物(wu)學實驗(yan)(yan)，包(bao)括酶切、PCR、測序、連接和(he)(he)轉(zhuan)(zhuan)化(hua)等試(shi)(shi)驗(yan)(yan)。本(ben)試(shi)(shi)劑(ji)(ji)盒無需(xu)使(shi)(shi)用(yong)(yong)(yong)酚等有(you)(you)(you)毒試(shi)(shi)劑(ji)(ji)，操(cao)作安全(quan)。操(cao)作步驟(zou)： 1、取(qu)(qu) 1-5ml 酵(jiao)(jiao)母(mu)(mu)培養物(wu)（不(bu)超過(guo) 5×107 cells），12000rpm 離(li)(li)(li)(li)(li)(li)心(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin) 1min，盡(jin)量(liang)吸(xi)(xi)除(chu)上(shang)清(qing)(qing)（菌(jun)液較多時(shi)可以通過(guo)多次(ci)離(li)(li)(li)(li)(li)(li)心(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)將(jiang)(jiang)菌(jun)體沉(chen)淀(dian)(dian)(dian)收集(ji)(ji)到(dao)(dao)一(yi)個離(li)(li)(li)(li)(li)(li)心(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)管(guan)中(zhong)(zhong)(zhong)）。 2、酵(jiao)(jiao)母(mu)(mu)細(xi)(xi)胞壁的(de)(de)(de)(de)破除(chu)： A、酶法(fa)：向(xiang)酵(jiao)(jiao)母(mu)(mu)菌(jun)體中(zhong)(zhong)(zhong)加(jia)(jia)入(ru)(ru)(ru)(ru)(ru)(ru) 470ul 山梨(li)醇 Buffer，充(chong)(chong)分(fen)懸(xuan)(xuan)浮(fu)(fu)菌(jun)體，加(jia)(jia)入(ru)(ru)(ru)(ru)(ru)(ru) 25ul 酵(jiao)(jiao)母(mu)(mu)破壁酶和(he)(he) 5ul 巰基(ji)還原劑(ji)(ji)，充(chong)(chong)分(fen)混(hun)勻，30℃處(chu)理 1-2h，期間(jian)可顛倒(dao)離(li)(li)(li)(li)(li)(li)心(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)管(guan)混(hun)勻數次(ci)。12000rpm 離(li)(li)(li)(li)(li)(li)心(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin) 1min，棄(qi)(qi)上(shang)清(qing)(qing)，收集(ji)(ji)沉(chen)淀(dian)(dian)(dian)。向(xiang)沉(chen)淀(dian)(dian)(dian)中(zhong)(zhong)(zhong)加(jia)(jia)入(ru)(ru)(ru)(ru)(ru)(ru) 250ulYP1（請(qing)(qing)先檢查是(shi)否已加(jia)(jia)入(ru)(ru)(ru)(ru)(ru)(ru) RNaseA），充(chong)(chong)分(fen)懸(xuan)(xuan)浮(fu)(fu)沉(chen)淀(dian)(dian)(dian)。注(zhu)意(yi)(yi)：如(ru)果(guo)菌(jun)塊未(wei)*混(hun)勻，會影響裂解導致質(zhi)(zhi)(zhi)粒(li)(li)提取(qu)(qu)量(liang)和(he)(he)純(chun)度偏低(di)。  B、玻(bo)璃(li)珠法(fa)：向(xiang)酵(jiao)(jiao)母(mu)(mu)菌(jun)體中(zhong)(zhong)(zhong)加(jia)(jia)入(ru)(ru)(ru)(ru)(ru)(ru) 250ul YP 1（請(qing)(qing)先檢查是(shi)否已加(jia)(jia)入(ru)(ru)(ru)(ru)(ru)(ru) RNaseA），充(chong)(chong)分(fen)懸(xuan)(xuan)浮(fu)(fu)沉(chen)淀(dian)(dian)(dian)。加(jia)(jia)入(ru)(ru)(ru)(ru)(ru)(ru) 150-200ul 酸洗(xi)玻(bo)璃(li)珠（自備(bei)），漩渦(wo)振蕩 10min。簡(jian)短離(li)(li)(li)(li)(li)(li)心(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)使(shi)(shi)玻(bo)璃(li)珠沉(chen)降到(dao)(dao)離(li)(li)(li)(li)(li)(li)心(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)管(guan)底，吸(xi)(xi)取(qu)(qu)上(shang)清(qing)(qing)（如(ru)上(shang)清(qing)(qing)有(you)(you)(you)所(suo)損失，請(qing)(qing)用(yong)(yong)(yong) YP 1 補足(zu) 250ul）于(yu)另一(yi)干凈離(li)(li)(li)(li)(li)(li)心(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)管(guan)中(zhong)(zhong)(zhong)。 3、向(xiang)離(li)(li)(li)(li)(li)(li)心(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)管(guan)中(zhong)(zhong)(zhong)加(jia)(jia)入(ru)(ru)(ru)(ru)(ru)(ru) 250ul YP2，溫(wen)和(he)(he)地(di)上(shang)下翻轉(zhuan)(zhuan) 6-8 次(ci)使(shi)(shi)菌(jun)體充(chong)(chong)分(fen)裂解。注(zhu)意(yi)(yi)：混(hun)勻一(yi)定要(yao)溫(wen)和(he)(he)，以免(mian)污染(ran)細(xi)(xi)菌(jun)基(ji)因組 DNA，此(ci)時(shi)菌(jun)液應變(bian)得(de)清(qing)(qing)亮粘稠(chou)，作用(yong)(yong)(yong)時(shi)間(jian)不(bu)要(yao)超過(guo) 5 min，以免(mian)質(zhi)(zhi)(zhi)粒(li)(li)受到(dao)(dao)破壞。 4、向(xiang)離(li)(li)(li)(li)(li)(li)心(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)管(guan)中(zhong)(zhong)(zhong)加(jia)(jia)入(ru)(ru)(ru)(ru)(ru)(ru) 350ul YP3，立即(ji)溫(wen)和(he)(he)地(di)上(shang)下翻轉(zhuan)(zhuan) 6-8 次(ci)，充(chong)(chong)分(fen)混(hun)勻，此(ci)時(shi)會出現白(bai)色絮狀沉(chen)淀(dian)(dian)(dian)。12000rpm 離(li)(li)(li)(li)(li)(li)心(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin) 10 min，用(yong)(yong)(yong)移液器小(xiao)(xiao)心(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)地(di)將(jiang)(jiang)上(shang)清(qing)(qing)轉(zhuan)(zhuan)移到(dao)(dao)另一(yi)個干凈的(de)(de)(de)(de)離(li)(li)(li)(li)(li)(li)心(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)管(guan)中(zhong)(zhong)(zhong)，盡(jin)量(liang)不(bu)要(yao)吸(xi)(xi)出沉(chen)淀(dian)(dian)(dian)。注(zhu)意(yi)(yi)：YP3 加(jia)(jia)入(ru)(ru)(ru)(ru)(ru)(ru)后應立即(ji)混(hun)合，避(bi)免(mian)產生局部沉(chen)淀(dian)(dian)(dian)。如(ru)果(guo)上(shang)清(qing)(qing)中(zhong)(zhong)(zhong)還有(you)(you)(you)微小(xiao)(xiao)白(bai)色沉(chen)淀(dian)(dian)(dian)，可再次(ci)離(li)(li)(li)(li)(li)(li)心(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)后取(qu)(qu)上(shang)清(qing)(qing)。 5、將(jiang)(jiang)上(shang)一(yi)步所(suo)得(de)上(shang)清(qing)(qing)液加(jia)(jia)入(ru)(ru)(ru)(ru)(ru)(ru)吸(xi)(xi)附(fu)(fu)(fu)(fu)(fu)柱(zhu)(zhu)(zhu)(zhu)(zhu)中(zhong)(zhong)(zhong)，室溫(wen)放(fang)(fang)(fang)置(zhi)(zhi) 2min，12000rpm 離(li)(li)(li)(li)(li)(li)心(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin) 1min，倒(dao)掉收集(ji)(ji)管(guan)中(zhong)(zhong)(zhong)的(de)(de)(de)(de)廢液，將(jiang)(jiang)吸(xi)(xi)附(fu)(fu)(fu)(fu)(fu)柱(zhu)(zhu)(zhu)(zhu)(zhu)重(zhong)新(xin)放(fang)(fang)(fang)回收集(ji)(ji)管(guan)中(zhong)(zhong)(zhong)。 6、向(xiang)吸(xi)(xi)附(fu)(fu)(fu)(fu)(fu)柱(zhu)(zhu)(zhu)(zhu)(zhu)中(zhong)(zhong)(zhong)加(jia)(jia)入(ru)(ru)(ru)(ru)(ru)(ru) 600ul 漂洗(xi)液(使(shi)(shi)用(yong)(yong)(yong)前(qian)請(qing)(qing)先檢查是(shi)否已加(jia)(jia)入(ru)(ru)(ru)(ru)(ru)(ru)無水(shui)(shui)乙(yi)醇)，12000rpm 離(li)(li)(li)(li)(li)(li)心(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin) 1min，棄(qi)(qi)廢液，將(jiang)(jiang)吸(xi)(xi)附(fu)(fu)(fu)(fu)(fu)柱(zhu)(zhu)(zhu)(zhu)(zhu)放(fang)(fang)(fang)入(ru)(ru)(ru)(ru)(ru)(ru)收集(ji)(ji)管(guan)中(zhong)(zhong)(zhong)。 7、向(xiang)吸(xi)(xi)附(fu)(fu)(fu)(fu)(fu)柱(zhu)(zhu)(zhu)(zhu)(zhu)中(zhong)(zhong)(zhong)加(jia)(jia)入(ru)(ru)(ru)(ru)(ru)(ru) 600ul 漂洗(xi)液，12000rpm 離(li)(li)(li)(li)(li)(li)心(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin) 1min，棄(qi)(qi)廢液，將(jiang)(jiang)吸(xi)(xi)附(fu)(fu)(fu)(fu)(fu)柱(zhu)(zhu)(zhu)(zhu)(zhu)放(fang)(fang)(fang)入(ru)(ru)(ru)(ru)(ru)(ru)收集(ji)(ji)管(guan)中(zhong)(zhong)(zhong)。 8、12000rpm 離(li)(li)(li)(li)(li)(li)心(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin) 2min，將(jiang)(jiang)吸(xi)(xi)附(fu)(fu)(fu)(fu)(fu)柱(zhu)(zhu)(zhu)(zhu)(zhu)敞口置(zhi)(zhi)于(yu)室溫(wen)或(huo) 50℃溫(wen)箱放(fang)(fang)(fang)置(zhi)(zhi)數分(fen)鐘，目的(de)(de)(de)(de)是(shi)將(jiang)(jiang)吸(xi)(xi)附(fu)(fu)(fu)(fu)(fu)柱(zhu)(zhu)(zhu)(zhu)(zhu)中(zhong)(zhong)(zhong)殘余的(de)(de)(de)(de)漂洗(xi)液去(qu)除(chu)，否則漂洗(xi)液中(zhong)(zhong)(zhong)的(de)(de)(de)(de)乙(yi)醇會影響后續的(de)(de)(de)(de)實驗(yan)(yan)如(ru)酶切、PCR 等。 9、將(jiang)(jiang)吸(xi)(xi)附(fu)(fu)(fu)(fu)(fu)柱(zhu)(zhu)(zhu)(zhu)(zhu)放(fang)(fang)(fang)入(ru)(ru)(ru)(ru)(ru)(ru)一(yi)個干凈的(de)(de)(de)(de)離(li)(li)(li)(li)(li)(li)心(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)管(guan)中(zhong)(zhong)(zhong)，向(xiang)吸(xi)(xi)附(fu)(fu)(fu)(fu)(fu)膜中(zhong)(zhong)(zhong)央懸(xuan)(xuan)空滴加(jia)(jia) 50-200ul 經 65℃水(shui)(shui)浴(yu)預熱的(de)(de)(de)(de)洗(xi)脫液，室溫(wen)放(fang)(fang)(fang)置(zhi)(zhi) 2min， 12000rpm 離(li)(li)(li)(li)(li)(li)心(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin) 1min。 10、為了增加(jia)(jia)質(zhi)(zhi)(zhi)粒(li)(li)的(de)(de)(de)(de)回收效(xiao)率，可將(jiang)(jiang)得(de)到(dao)(dao)的(de)(de)(de)(de)洗(xi)脫液重(zhong)新(xin)加(jia)(jia)入(ru)(ru)(ru)(ru)(ru)(ru)吸(xi)(xi)附(fu)(fu)(fu)(fu)(fu)柱(zhu)(zhu)(zhu)(zhu)(zhu)中(zhong)(zhong)(zhong)，室溫(wen)放(fang)(fang)(fang)置(zhi)(zhi) 2min，12000rpm 離(li)(li)(li)(li)(li)(li)心(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin)(xin) 1min。

Solarbio-酵母質粒小量提取試劑盒

注意事項：

1、使(shi)用前(qian)請(qing)先檢(jian)(jian)(jian)查 YP2 和 YP3 是否(fou)出現混(hun)濁，如(ru)有(you)混(hun)濁現象(xiang)，可在(zai)(zai)(zai) 37℃水(shui)(shui)浴中加熱(re)幾(ji)分鐘，待溶液(ye)(ye)恢復澄清后(hou)再使(shi)用。 2、洗(xi)脫緩(huan)沖液(ye)(ye)體(ti)積不應(ying)(ying)少于 50ul，體(ti)積過小(xiao)影響回收(shou)(shou)效(xiao)率(lv)；洗(xi)脫液(ye)(ye)的 pH 值(zhi)(zhi)(zhi)對洗(xi)脫效(xiao)率(lv)也有(you)影響，若需要用水(shui)(shui)做洗(xi)脫液(ye)(ye)應(ying)(ying)保證其 pH 值(zhi)(zhi)(zhi)在(zai)(zai)(zai) 8.0 左右(you)(可用 NaOH 將水(shui)(shui)的 pH 值(zhi)(zhi)(zhi)調此范圍)，pH 值(zhi)(zhi)(zhi)低于 7.0 會降低洗(xi)脫效(xiao)率(lv)；DNA 產(chan)物應(ying)(ying)保存在(zai)(zai)(zai)-20℃，以(yi)防 DNA 降解。 3、質粒得(de)(de)率(lv)與(yu)(yu)酵(jiao)母菌株(zhu)、質粒拷(kao)貝(bei)數(shu)、培養條(tiao)件等因(yin)素有(you)關(guan)(guan)。通常(chang)酵(jiao)母質粒拷(kao)貝(bei)數(shu)都(dou)很(hen)低，一(yi)般通過電泳(yong)或分光光度(du)(du)計(ji)法都(dou)很(hen)難檢(jian)(jian)(jian)測(ce)(ce)到，可通過 PCR 或轉化大(da)腸桿(gan)菌來檢(jian)(jian)(jian)測(ce)(ce)。 4、DNA 濃度(du)(du)及純度(du)(du)檢(jian)(jian)(jian)測(ce)(ce)：得(de)(de)到的基因(yin)組(zu) DNA 片段的大(da)小(xiao)與(yu)(yu)樣品保存時(shi)間、操作過程(cheng)中的剪切力等因(yin)素有(you)關(guan)(guan)。回收(shou)(shou)得(de)(de)到的 DNA 片段可用瓊脂糖凝(ning)膠電泳(yong)和紫外分光光度(du)(du)計(ji)檢(jian)(jian)(jian)測(ce)(ce)濃度(du)(du)與(yu)(yu)純度(du)(du)。DNA 應(ying)(ying)在(zai)(zai)(zai) OD260 處(chu)有(you)顯(xian)著吸收(shou)(shou)峰， OD260 值(zhi)(zhi)(zhi)為 1 相當(dang)于大(da)約 50 μg/ml 雙鏈 DNA、40 μg/ml 單鏈 DNA。OD260/OD280 比(bi)值(zhi)(zhi)(zhi)應(ying)(ying)為 1.7-1.9，如(ru)果洗(xi)脫時(shi)不使(shi)用洗(xi)脫緩(huan)沖液(ye)(ye)，而使(shi)用去離(li)子(zi)水(shui)(shui)，比(bi)值(zhi)(zhi)(zhi)會偏低，因(yin)為 pH 值(zhi)(zhi)(zhi)和離(li)子(zi)存在(zai)(zai)(zai)會影響光吸收(shou)(shou)值(zhi)(zhi)(zhi)，但并(bing)不表示純度(du)(du)低。