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Solarbio-酵母質粒小量提取試劑盒 

產品簡(jian)(jian)介：試(shi)劑盒(he)采用(yong)(yong)(yong)(yong)酶(mei)法破(po)碎(sui)酵(jiao)母(mu)(mu)(mu)細(xi)(xi)胞壁和堿裂解(jie)法裂解(jie)酵(jiao)母(mu)(mu)(mu)細(xi)(xi)胞來提(ti)取酵(jiao)母(mu)(mu)(mu)質(zhi)(zhi)粒(li) DNA。所(suo)采用(yong)(yong)(yong)(yong)的(de)(de)(de)(de)酵(jiao)母(mu)(mu)(mu)破(po)壁酶(mei)能(neng)有(you)效(xiao)地破(po)壞酵(jiao)母(mu)(mu)(mu)細(xi)(xi)胞壁，提(ti)高酵(jiao)母(mu)(mu)(mu)質(zhi)(zhi)粒(li) DNA 的(de)(de)(de)(de)產量。吸(xi)(xi)(xi)(xi)(xi)(xi)(xi)附(fu)柱(zhu)(zhu)(zhu)中(zhong)(zhong)(zhong)(zhong)(zhong)采用(yong)(yong)(yong)(yong)的(de)(de)(de)(de)硅基質(zhi)(zhi)材料能(neng)高效(xiao)、專一地吸(xi)(xi)(xi)(xi)(xi)(xi)(xi)附(fu) DNA，可(ke)(ke)限(xian)度去除(chu)(chu)雜質(zhi)(zhi)蛋白質(zhi)(zhi)及細(xi)(xi)胞中(zhong)(zhong)(zhong)(zhong)(zhong)其他有(you)機(ji)化合(he)物(wu)。使(shi)用(yong)(yong)(yong)(yong)本試(shi)劑盒(he)提(ti)取的(de)(de)(de)(de)酵(jiao)母(mu)(mu)(mu)質(zhi)(zhi)粒(li) DNA 可(ke)(ke)適用(yong)(yong)(yong)(yong)于(yu)各種常規的(de)(de)(de)(de)分子生(sheng)物(wu)學實驗(yan)，包括酶(mei)切、PCR、測序、連接和轉化等試(shi)驗(yan)。本試(shi)劑盒(he)無(wu)需使(shi)用(yong)(yong)(yong)(yong)酚(fen)等有(you)毒試(shi)劑，操作(zuo)(zuo)安全。操作(zuo)(zuo)步驟： 1、取 1-5ml 酵(jiao)母(mu)(mu)(mu)培(pei)養物(wu)（不(bu)超過(guo)(guo) 5×107 cells），12000rpm 離(li)(li)(li)(li)(li)(li)心 1min，盡量吸(xi)(xi)(xi)(xi)(xi)(xi)(xi)除(chu)(chu)上(shang)清(qing)(qing)（菌(jun)(jun)(jun)液(ye)(ye)較多(duo)時可(ke)(ke)以通過(guo)(guo)多(duo)次(ci)離(li)(li)(li)(li)(li)(li)心將(jiang)(jiang)(jiang)(jiang)菌(jun)(jun)(jun)體(ti)沉(chen)淀(dian)收(shou)集(ji)(ji)到(dao)(dao)(dao)一個(ge)離(li)(li)(li)(li)(li)(li)心管(guan)(guan)(guan)(guan)(guan)(guan)中(zhong)(zhong)(zhong)(zhong)(zhong)）。 2、酵(jiao)母(mu)(mu)(mu)細(xi)(xi)胞壁的(de)(de)(de)(de)破(po)除(chu)(chu)： A、酶(mei)法：向(xiang)(xiang)酵(jiao)母(mu)(mu)(mu)菌(jun)(jun)(jun)體(ti)中(zhong)(zhong)(zhong)(zhong)(zhong)加(jia)(jia)(jia)(jia)(jia)(jia)入(ru)(ru)(ru)(ru)(ru)(ru) 470ul 山梨醇 Buffer，充(chong)分懸浮(fu)(fu)(fu)菌(jun)(jun)(jun)體(ti)，加(jia)(jia)(jia)(jia)(jia)(jia)入(ru)(ru)(ru)(ru)(ru)(ru) 25ul 酵(jiao)母(mu)(mu)(mu)破(po)壁酶(mei)和 5ul 巰(qiu)基還原劑，充(chong)分混(hun)勻(yun)，30℃處理 1-2h，期間可(ke)(ke)顛倒離(li)(li)(li)(li)(li)(li)心管(guan)(guan)(guan)(guan)(guan)(guan)混(hun)勻(yun)數次(ci)。12000rpm 離(li)(li)(li)(li)(li)(li)心 1min，棄(qi)上(shang)清(qing)(qing)，收(shou)集(ji)(ji)沉(chen)淀(dian)。向(xiang)(xiang)沉(chen)淀(dian)中(zhong)(zhong)(zhong)(zhong)(zhong)加(jia)(jia)(jia)(jia)(jia)(jia)入(ru)(ru)(ru)(ru)(ru)(ru) 250ulYP1（請(qing)先檢(jian)查(cha)(cha)是(shi)否(fou)已(yi)(yi)加(jia)(jia)(jia)(jia)(jia)(jia)入(ru)(ru)(ru)(ru)(ru)(ru) RNaseA），充(chong)分懸浮(fu)(fu)(fu)沉(chen)淀(dian)。注(zhu)意(yi)(yi)：如(ru)果菌(jun)(jun)(jun)塊未*混(hun)勻(yun)，會(hui)影響裂解(jie)導致質(zhi)(zhi)粒(li)提(ti)取量和純度偏低。  B、玻(bo)(bo)璃珠(zhu)法：向(xiang)(xiang)酵(jiao)母(mu)(mu)(mu)菌(jun)(jun)(jun)體(ti)中(zhong)(zhong)(zhong)(zhong)(zhong)加(jia)(jia)(jia)(jia)(jia)(jia)入(ru)(ru)(ru)(ru)(ru)(ru) 250ul YP 1（請(qing)先檢(jian)查(cha)(cha)是(shi)否(fou)已(yi)(yi)加(jia)(jia)(jia)(jia)(jia)(jia)入(ru)(ru)(ru)(ru)(ru)(ru) RNaseA），充(chong)分懸浮(fu)(fu)(fu)沉(chen)淀(dian)。加(jia)(jia)(jia)(jia)(jia)(jia)入(ru)(ru)(ru)(ru)(ru)(ru) 150-200ul 酸洗(xi)玻(bo)(bo)璃珠(zhu)（自備），漩渦振蕩 10min。簡(jian)(jian)短離(li)(li)(li)(li)(li)(li)心使(shi)玻(bo)(bo)璃珠(zhu)沉(chen)降到(dao)(dao)(dao)離(li)(li)(li)(li)(li)(li)心管(guan)(guan)(guan)(guan)(guan)(guan)底(di)，吸(xi)(xi)(xi)(xi)(xi)(xi)(xi)取上(shang)清(qing)(qing)（如(ru)上(shang)清(qing)(qing)有(you)所(suo)損失，請(qing)用(yong)(yong)(yong)(yong) YP 1 補足 250ul）于(yu)另一干凈(jing)離(li)(li)(li)(li)(li)(li)心管(guan)(guan)(guan)(guan)(guan)(guan)中(zhong)(zhong)(zhong)(zhong)(zhong)。 3、向(xiang)(xiang)離(li)(li)(li)(li)(li)(li)心管(guan)(guan)(guan)(guan)(guan)(guan)中(zhong)(zhong)(zhong)(zhong)(zhong)加(jia)(jia)(jia)(jia)(jia)(jia)入(ru)(ru)(ru)(ru)(ru)(ru) 250ul YP2，溫(wen)(wen)(wen)和地上(shang)下(xia)(xia)翻(fan)轉 6-8 次(ci)使(shi)菌(jun)(jun)(jun)體(ti)充(chong)分裂解(jie)。注(zhu)意(yi)(yi)：混(hun)勻(yun)一定要(yao)溫(wen)(wen)(wen)和，以免污(wu)染細(xi)(xi)菌(jun)(jun)(jun)基因組 DNA，此時菌(jun)(jun)(jun)液(ye)(ye)應變得清(qing)(qing)亮粘稠，作(zuo)(zuo)用(yong)(yong)(yong)(yong)時間不(bu)要(yao)超過(guo)(guo) 5 min，以免質(zhi)(zhi)粒(li)受到(dao)(dao)(dao)破(po)壞。 4、向(xiang)(xiang)離(li)(li)(li)(li)(li)(li)心管(guan)(guan)(guan)(guan)(guan)(guan)中(zhong)(zhong)(zhong)(zhong)(zhong)加(jia)(jia)(jia)(jia)(jia)(jia)入(ru)(ru)(ru)(ru)(ru)(ru) 350ul YP3，立(li)即溫(wen)(wen)(wen)和地上(shang)下(xia)(xia)翻(fan)轉 6-8 次(ci)，充(chong)分混(hun)勻(yun)，此時會(hui)出(chu)現白色(se)絮狀沉(chen)淀(dian)。12000rpm 離(li)(li)(li)(li)(li)(li)心 10 min，用(yong)(yong)(yong)(yong)移(yi)液(ye)(ye)器小心地將(jiang)(jiang)(jiang)(jiang)上(shang)清(qing)(qing)轉移(yi)到(dao)(dao)(dao)另一個(ge)干凈(jing)的(de)(de)(de)(de)離(li)(li)(li)(li)(li)(li)心管(guan)(guan)(guan)(guan)(guan)(guan)中(zhong)(zhong)(zhong)(zhong)(zhong)，盡量不(bu)要(yao)吸(xi)(xi)(xi)(xi)(xi)(xi)(xi)出(chu)沉(chen)淀(dian)。注(zhu)意(yi)(yi)：YP3 加(jia)(jia)(jia)(jia)(jia)(jia)入(ru)(ru)(ru)(ru)(ru)(ru)后應立(li)即混(hun)合(he)，避免產生(sheng)局(ju)部沉(chen)淀(dian)。如(ru)果上(shang)清(qing)(qing)中(zhong)(zhong)(zhong)(zhong)(zhong)還有(you)微小白色(se)沉(chen)淀(dian)，可(ke)(ke)再次(ci)離(li)(li)(li)(li)(li)(li)心后取上(shang)清(qing)(qing)。 5、將(jiang)(jiang)(jiang)(jiang)上(shang)一步所(suo)得上(shang)清(qing)(qing)液(ye)(ye)加(jia)(jia)(jia)(jia)(jia)(jia)入(ru)(ru)(ru)(ru)(ru)(ru)吸(xi)(xi)(xi)(xi)(xi)(xi)(xi)附(fu)柱(zhu)(zhu)(zhu)中(zhong)(zhong)(zhong)(zhong)(zhong)，室(shi)溫(wen)(wen)(wen)放(fang)置 2min，12000rpm 離(li)(li)(li)(li)(li)(li)心 1min，倒掉收(shou)集(ji)(ji)管(guan)(guan)(guan)(guan)(guan)(guan)中(zhong)(zhong)(zhong)(zhong)(zhong)的(de)(de)(de)(de)廢液(ye)(ye)，將(jiang)(jiang)(jiang)(jiang)吸(xi)(xi)(xi)(xi)(xi)(xi)(xi)附(fu)柱(zhu)(zhu)(zhu)重新(xin)放(fang)回收(shou)集(ji)(ji)管(guan)(guan)(guan)(guan)(guan)(guan)中(zhong)(zhong)(zhong)(zhong)(zhong)。 6、向(xiang)(xiang)吸(xi)(xi)(xi)(xi)(xi)(xi)(xi)附(fu)柱(zhu)(zhu)(zhu)中(zhong)(zhong)(zhong)(zhong)(zhong)加(jia)(jia)(jia)(jia)(jia)(jia)入(ru)(ru)(ru)(ru)(ru)(ru) 600ul 漂(piao)洗(xi)液(ye)(ye)(使(shi)用(yong)(yong)(yong)(yong)前(qian)請(qing)先檢(jian)查(cha)(cha)是(shi)否(fou)已(yi)(yi)加(jia)(jia)(jia)(jia)(jia)(jia)入(ru)(ru)(ru)(ru)(ru)(ru)無(wu)水(shui)乙醇)，12000rpm 離(li)(li)(li)(li)(li)(li)心 1min，棄(qi)廢液(ye)(ye)，將(jiang)(jiang)(jiang)(jiang)吸(xi)(xi)(xi)(xi)(xi)(xi)(xi)附(fu)柱(zhu)(zhu)(zhu)放(fang)入(ru)(ru)(ru)(ru)(ru)(ru)收(shou)集(ji)(ji)管(guan)(guan)(guan)(guan)(guan)(guan)中(zhong)(zhong)(zhong)(zhong)(zhong)。 7、向(xiang)(xiang)吸(xi)(xi)(xi)(xi)(xi)(xi)(xi)附(fu)柱(zhu)(zhu)(zhu)中(zhong)(zhong)(zhong)(zhong)(zhong)加(jia)(jia)(jia)(jia)(jia)(jia)入(ru)(ru)(ru)(ru)(ru)(ru) 600ul 漂(piao)洗(xi)液(ye)(ye)，12000rpm 離(li)(li)(li)(li)(li)(li)心 1min，棄(qi)廢液(ye)(ye)，將(jiang)(jiang)(jiang)(jiang)吸(xi)(xi)(xi)(xi)(xi)(xi)(xi)附(fu)柱(zhu)(zhu)(zhu)放(fang)入(ru)(ru)(ru)(ru)(ru)(ru)收(shou)集(ji)(ji)管(guan)(guan)(guan)(guan)(guan)(guan)中(zhong)(zhong)(zhong)(zhong)(zhong)。 8、12000rpm 離(li)(li)(li)(li)(li)(li)心 2min，將(jiang)(jiang)(jiang)(jiang)吸(xi)(xi)(xi)(xi)(xi)(xi)(xi)附(fu)柱(zhu)(zhu)(zhu)敞口置于(yu)室(shi)溫(wen)(wen)(wen)或 50℃溫(wen)(wen)(wen)箱(xiang)放(fang)置數分鐘，目的(de)(de)(de)(de)是(shi)將(jiang)(jiang)(jiang)(jiang)吸(xi)(xi)(xi)(xi)(xi)(xi)(xi)附(fu)柱(zhu)(zhu)(zhu)中(zhong)(zhong)(zhong)(zhong)(zhong)殘余的(de)(de)(de)(de)漂(piao)洗(xi)液(ye)(ye)去除(chu)(chu)，否(fou)則漂(piao)洗(xi)液(ye)(ye)中(zhong)(zhong)(zhong)(zhong)(zhong)的(de)(de)(de)(de)乙醇會(hui)影響后續的(de)(de)(de)(de)實驗(yan)如(ru)酶(mei)切、PCR 等。 9、將(jiang)(jiang)(jiang)(jiang)吸(xi)(xi)(xi)(xi)(xi)(xi)(xi)附(fu)柱(zhu)(zhu)(zhu)放(fang)入(ru)(ru)(ru)(ru)(ru)(ru)一個(ge)干凈(jing)的(de)(de)(de)(de)離(li)(li)(li)(li)(li)(li)心管(guan)(guan)(guan)(guan)(guan)(guan)中(zhong)(zhong)(zhong)(zhong)(zhong)，向(xiang)(xiang)吸(xi)(xi)(xi)(xi)(xi)(xi)(xi)附(fu)膜中(zhong)(zhong)(zhong)(zhong)(zhong)央懸空滴(di)加(jia)(jia)(jia)(jia)(jia)(jia) 50-200ul 經(jing) 65℃水(shui)浴(yu)預熱的(de)(de)(de)(de)洗(xi)脫(tuo)液(ye)(ye)，室(shi)溫(wen)(wen)(wen)放(fang)置 2min， 12000rpm 離(li)(li)(li)(li)(li)(li)心 1min。 10、為了增(zeng)加(jia)(jia)(jia)(jia)(jia)(jia)質(zhi)(zhi)粒(li)的(de)(de)(de)(de)回收(shou)效(xiao)率，可(ke)(ke)將(jiang)(jiang)(jiang)(jiang)得到(dao)(dao)(dao)的(de)(de)(de)(de)洗(xi)脫(tuo)液(ye)(ye)重新(xin)加(jia)(jia)(jia)(jia)(jia)(jia)入(ru)(ru)(ru)(ru)(ru)(ru)吸(xi)(xi)(xi)(xi)(xi)(xi)(xi)附(fu)柱(zhu)(zhu)(zhu)中(zhong)(zhong)(zhong)(zhong)(zhong)，室(shi)溫(wen)(wen)(wen)放(fang)置 2min，12000rpm 離(li)(li)(li)(li)(li)(li)心 1min。

Solarbio-酵母質粒小量提取試劑盒

注意事項：

1、使用前請先檢(jian)(jian)查(cha) YP2 和 YP3 是否(fou)出現(xian)混濁(zhuo)，如有(you)混濁(zhuo)現(xian)象(xiang)，可(ke)(ke)在 37℃水浴中加熱幾分(fen)鐘，待溶液恢復澄清(qing)后再使用。 2、洗(xi)(xi)(xi)脫(tuo)(tuo)(tuo)緩(huan)沖液體(ti)積(ji)不(bu)應(ying)少于(yu) 50ul，體(ti)積(ji)過(guo)(guo)小(xiao)影響(xiang)回(hui)收(shou)(shou)效(xiao)率；洗(xi)(xi)(xi)脫(tuo)(tuo)(tuo)液的(de) pH 值(zhi)(zhi)對洗(xi)(xi)(xi)脫(tuo)(tuo)(tuo)效(xiao)率也有(you)影響(xiang)，若需要用水做洗(xi)(xi)(xi)脫(tuo)(tuo)(tuo)液應(ying)保證(zheng)其(qi) pH 值(zhi)(zhi)在 8.0 左右(可(ke)(ke)用 NaOH 將(jiang)水的(de) pH 值(zhi)(zhi)調此(ci)范圍)，pH 值(zhi)(zhi)低(di)于(yu) 7.0 會降低(di)洗(xi)(xi)(xi)脫(tuo)(tuo)(tuo)效(xiao)率；DNA 產(chan)物應(ying)保存(cun)在-20℃，以防(fang) DNA 降解。 3、質(zhi)粒得率與(yu)酵母(mu)菌(jun)株、質(zhi)粒拷(kao)貝數、培(pei)養(yang)條件(jian)等因(yin)素(su)有(you)關(guan)。通(tong)常酵母(mu)質(zhi)粒拷(kao)貝數都很低(di)，一般通(tong)過(guo)(guo)電(dian)泳或分(fen)光光度(du)(du)計法都很難檢(jian)(jian)測(ce)到，可(ke)(ke)通(tong)過(guo)(guo) PCR 或轉化大腸桿菌(jun)來檢(jian)(jian)測(ce)。 4、DNA 濃度(du)(du)及純度(du)(du)檢(jian)(jian)測(ce)：得到的(de)基因(yin)組 DNA 片段的(de)大小(xiao)與(yu)樣品保存(cun)時間、操(cao)作過(guo)(guo)程中的(de)剪切力等因(yin)素(su)有(you)關(guan)。回(hui)收(shou)(shou)得到的(de) DNA 片段可(ke)(ke)用瓊脂糖凝膠(jiao)電(dian)泳和紫(zi)外分(fen)光光度(du)(du)計檢(jian)(jian)測(ce)濃度(du)(du)與(yu)純度(du)(du)。DNA 應(ying)在 OD260 處(chu)有(you)顯著吸(xi)收(shou)(shou)峰， OD260 值(zhi)(zhi)為 1 相當于(yu)大約(yue) 50 μg/ml 雙(shuang)鏈(lian) DNA、40 μg/ml 單(dan)鏈(lian) DNA。OD260/OD280 比值(zhi)(zhi)應(ying)為 1.7-1.9，如果洗(xi)(xi)(xi)脫(tuo)(tuo)(tuo)時不(bu)使用洗(xi)(xi)(xi)脫(tuo)(tuo)(tuo)緩(huan)沖液，而使用去(qu)離子水，比值(zhi)(zhi)會偏(pian)低(di)，因(yin)為 pH 值(zhi)(zhi)和離子存(cun)在會影響(xiang)光吸(xi)收(shou)(shou)值(zhi)(zhi)，但并不(bu)表示(shi)純度(du)(du)低(di)。