材料：質(zhi)粒DNA指數生長的(de)(de)(de)(de)真核細(xi)(xi)胞(bao)(bao)(bao)PEI（聚乙烯亞胺）1×HBS（pH7.4）配方：PEI儲存(cun)液(ye)（100μM）：稱取125mgPEI粉末溶解(jie)于(yu)(yu)50ml1×HBS（pH7.4）中，0.2μm濾(lv)膜過濾(lv)，儲存(cun)于(yu)(yu)4℃備(bei)用(yong)。1×HBS（pH7.4）：將8.76gNaCl溶解(jie)于(yu)(yu)900ml超純(chun)水，加(jia)入20ml1M的(de)(de)(de)(de)HEPES，調pH值到7.4，定容至1L，過濾(lv)（0.2μm濾(lv)膜）后(hou)儲存(cun)于(yu)(yu)4℃備(bei)用(yong)。方法：1．細(xi)(xi)胞(bao)(bao)(bao)分盤：通(tong)過胰酶消化收集細(xi)(xi)胞(bao)(bao)(bao)，用(yong)適當的(de)(de)(de)(de)完全培養基(ji)以4×105至8×105細(xi)(xi)胞(bao)(bao)(bao)/cm2的(de)(de)(de)(de)密度平鋪細(xi)(xi)胞(bao)(bao)(bao)于(yu)(yu)60mm組織培養皿(min)(min)上(shang)（根(gen)據實驗需要選擇培養皿(min)(min)，使細(xi)(xi)胞(bao)(bao)(bao)貼壁后(hou)所占總面積達(da)到培養皿(min)(min)面積的(de)(de)(de)(de)70-90％）。根(gen)據細(xi)(xi)胞(bao)(bao)(bao)貼壁情況于(yu)(yu)含5％CO2的(de)(de)(de)(de)37℃溫箱中孵育8-24h,當細(xi)(xi)胞(bao)(bao)(bao)貼壁完全后(hou)即可開始(shi)轉(zhuan)(zhuan)(zhuan)(zhuan)染(ran)。轉(zhuan)(zhuan)(zhuan)(zhuan)染(ran)前換入2mL預(yu)熱的(de)(de)(de)(de)無血清培養基(ji)。更多關于(yu)(yu)PEI轉(zhuan)(zhuan)(zhuan)(zhuan)染(ran)試劑相(xiang)關產品(pin)技術(shu)問題，歡迎咨詢上(shang)海曼博生物。如想申請(qing)PEI試用(yong)裝，請(qing)關注我(wo)們的(de)(de)(de)(de)公眾號：Mine-bio，回復關鍵詞“PEI申請(qing)”，即可參加(jia)！哪個品(pin)牌的(de)(de)(de)(de)PEI轉(zhuan)(zhuan)(zhuan)(zhuan)染(ran)試劑質(zhi)量好？申請(qing)PEI轉(zhuan)(zhuan)(zhuan)(zhuan)染(ran)試劑試用(yong)裝轉(zhuan)(zhuan)(zhuan)(zhuan)染(ran)步驟

穩定(ding)轉(zhuan)(zhuan)(zhuan)染(ran)(ran)(ran)方法(fa)：1.接種(zhong)細(xi)胞：轉(zhuan)(zhuan)(zhuan)染(ran)(ran)(ran)前(qian)一(yi)(yi)(yi)晚，用(yong)(yong)膠原酶（WORTHINGTONG公(gong)司(si)LS004196)消化細(xi)胞并計(ji)數（不(bu)建議(yi)用(yong)(yong)胰酶）。調(diao)整細(xi)胞濃度，將細(xi)胞鋪(pu)入細(xi)胞培養(yang)的器皿(min)，每(mei)(mei)個孔置(zhi)入的細(xi)胞量應能(neng)使轉(zhuan)(zhuan)(zhuan)染(ran)(ran)(ran)時細(xi)胞匯合度達到70~80%。2.準備(bei)DNA-PEI復(fu)合物：DNA、PEI試(shi)(shi)(shi)劑(ji)(ji)和稀(xi)(xi)釋劑(ji)(ji)在進行以下步驟前(qian)需先使其(qi)升至(zhi)室溫(wen)。依(yi)據表(biao)1所示，用(yong)(yong)Opti-MEMITM（Invitrogen）或其(qi)他適(shi)合的無蛋白培養(yang)基稀(xi)(xi)釋適(shi)量DNA。用(yong)(yong)同樣(yang)的培養(yang)基稀(xi)(xi)釋PEI試(shi)(shi)(shi)劑(ji)(ji)。每(mei)(mei)1μgDNA需用(yong)(yong)1.5-5μL線性PEI轉(zhuan)(zhuan)(zhuan)染(ran)(ran)(ran)試(shi)(shi)(shi)劑(ji)(ji)(這個需要客戶自行摸索配比，每(mei)(mei)一(yi)(yi)(yi)批(pi)轉(zhuan)(zhuan)(zhuan)染(ran)(ran)(ran)試(shi)(shi)(shi)劑(ji)(ji)和每(mei)(mei)一(yi)(yi)(yi)批(pi)DNA比例(li)可(ke)能(neng)會稍有(you)不(bu)同）。一(yi)(yi)(yi)邊(bian)輕輕渦旋裝(zhuang)有(you)DNA溶液(ye)的試(shi)(shi)(shi)管(guan)，一(yi)(yi)(yi)邊(bian)將稀(xi)(xi)釋的線性PEI轉(zhuan)(zhuan)(zhuan)染(ran)(ran)(ran)試(shi)(shi)(shi)劑(ji)(ji)滴(di)加至(zhi)試(shi)(shi)(shi)管(guan)中(zhong)（注意：請(qing)(qing)勿顛倒添(tian)加順序）。充分混勻后，室溫(wen)靜(jing)置(zhi)10~25min以形成DNA-PEI復(fu)合物。當溶液(ye)體積較大時，請(qing)(qing)用(yong)(yong)圓底(di)聚丙烯管(guan)，例(li)如(ru)NEST5mL/14mL離心管(guan)。更(geng)(geng)多關(guan)于Polysciences PEI相關(guan)內容歡迎咨詢(xun)上海曼博生物！如(ru)想申請(qing)(qing)PEI試(shi)(shi)(shi)用(yong)(yong)裝(zhuang)，請(qing)(qing)關(guan)注我(wo)們(men)的公(gong)眾(zhong)號(hao)：Mine-bio，回(hui)復(fu)關(guan)鍵詞“PEI申請(qing)(qing)”，即(ji)可(ke)參加！?全球PEI轉(zhuan)(zhuan)(zhuan)染(ran)(ran)(ran)試(shi)(shi)(shi)劑(ji)(ji)試(shi)(shi)(shi)用(yong)(yong)裝(zhuang)幫助用(yong)(yong)戶更(geng)(geng)好(hao)地了解PEI轉(zhuan)(zhuan)(zhuan)染(ran)(ran)(ran)試(shi)(shi)(shi)劑(ji)(ji)的操作(zuo)流程(cheng)分子量為25000的線性PEI轉(zhuan)(zhuan)(zhuan)染(ran)(ran)(ran)試(shi)(shi)(shi)劑(ji)(ji)即(ji)Polysciences23966轉(zhuan)(zhuan)(zhuan)染(ran)(ran)(ran)效率比較高.

1.對(dui)于(yu)某些類(lei)型(xing)的(de)(de)(de)(de)細(xi)胞(bao)如HEK-293、HEK293T、NIH/3T3和COS細(xi)胞(bao)，在(zai)(zai)轉(zhuan)(zhuan)染(ran)前兩天鋪(pu)板(ban)可顯著提高(gao)轉(zhuan)(zhuan)入基因的(de)(de)(de)(de)表達水平。如果選擇轉(zhuan)(zhuan)染(ran)前兩天鋪(pu)板(ban)，可適當(dang)降低鋪(pu)板(ban)密度(du)，以(yi)確保轉(zhuan)(zhuan)染(ran)時細(xi)胞(bao)的(de)(de)(de)(de)匯合(he)度(du)仍(reng)(reng)為70~80%。2.對(dui)于(yu)接觸抑(yi)制敏感的(de)(de)(de)(de)細(xi)胞(bao)，可適當(dang)降低鋪(pu)板(ban)密度(du)。3.即(ji)使在(zai)(zai)有蛋(dan)(dan)白（如10%的(de)(de)(de)(de)血清(qing)）存(cun)在(zai)(zai)的(de)(de)(de)(de)情況下，DNA-PEI復(fu)合(he)物仍(reng)(reng)能轉(zhuan)(zhuan)染(ran)細(xi)胞(bao)，但是DNA-PEI復(fu)合(he)物必須在(zai)(zai)無蛋(dan)(dan)白存(cun)在(zai)(zai)的(de)(de)(de)(de)條件下形成(cheng)。我(wo)們(men)推薦使用(yong)(yong)Opti-MEMITM培(pei)養(yang)基以(yi)達到*轉(zhuan)(zhuan)染(ran)效率(lv)。其他(ta)的(de)(de)(de)(de)無蛋(dan)(dan)白培(pei)養(yang)基則需測試(shi)與線性(xing)PEI轉(zhuan)(zhuan)染(ran)試(shi)劑(ji)(ji)的(de)(de)(de)(de)兼容(rong)性(xing)。4.對(dui)大多數細(xi)胞(bao)來(lai)而言(yan)，每1μgDNA使用(yong)(yong)3.0μLEndoFectinTM試(shi)劑(ji)(ji)都(dou)能獲(huo)得較高(gao)轉(zhuan)(zhuan)染(ran)效率(lv)。使用(yong)(yong)者也(ye)可嘗(chang)試(shi)每1μgDNA使用(yong)(yong)1~4μL體積(ji)線性(xing)PEI轉(zhuan)(zhuan)染(ran)試(shi)劑(ji)(ji)進行優化。更(geng)多關(guan)于(yu)PEI轉(zhuan)(zhuan)染(ran)試(shi)劑(ji)(ji)相關(guan)產品技術問題，歡迎咨(zi)詢上(shang)海曼博生(sheng)物。?如想申請PEI試(shi)用(yong)(yong)裝(zhuang)，請關(guan)注(zhu)我(wo)們(men)的(de)(de)(de)(de)公眾(zhong)號：Mine-bio，回復(fu)關(guan)鍵詞“PEI申請”，即(ji)可參(can)加！

1.對于(yu)某些類型的(de)(de)細胞如(ru)(ru)HEK-293、HEK293T、NIH/3T3和COS細胞，在(zai)(zai)(zai)轉(zhuan)(zhuan)(zhuan)(zhuan)染前兩天(tian)鋪(pu)(pu)板可顯著提高(gao)轉(zhuan)(zhuan)(zhuan)(zhuan)入基(ji)因的(de)(de)表達水(shui)平。如(ru)(ru)果(guo)選擇轉(zhuan)(zhuan)(zhuan)(zhuan)染前兩天(tian)鋪(pu)(pu)板，可適當(dang)降低鋪(pu)(pu)板密(mi)度，以確保(bao)轉(zhuan)(zhuan)(zhuan)(zhuan)染時細胞的(de)(de)匯合(he)度仍為70~80%。2.對于(yu)接觸(chu)抑(yi)制(zhi)敏(min)感的(de)(de)細胞，可適當(dang)降低鋪(pu)(pu)板密(mi)度。3.即使(shi)在(zai)(zai)(zai)有(you)蛋(dan)(dan)白（如(ru)(ru)10%的(de)(de)血清(qing)）存在(zai)(zai)(zai)的(de)(de)情(qing)況(kuang)下(xia)，DNA-PEI復(fu)合(he)物(wu)(wu)仍能(neng)轉(zhuan)(zhuan)(zhuan)(zhuan)染細胞，但是DNA-PEI復(fu)合(he)物(wu)(wu)必須在(zai)(zai)(zai)無(wu)蛋(dan)(dan)白存在(zai)(zai)(zai)的(de)(de)條件(jian)下(xia)形成。我們推薦(jian)使(shi)用(yong)(yong)Opti-MEMITM培養基(ji)以達到*轉(zhuan)(zhuan)(zhuan)(zhuan)染效率(lv)。其他的(de)(de)無(wu)蛋(dan)(dan)白培養基(ji)則需測試(shi)(shi)與線性(xing)PEI轉(zhuan)(zhuan)(zhuan)(zhuan)染試(shi)(shi)劑的(de)(de)兼容性(xing)。4.對大多(duo)數細胞來而言(yan)，每(mei)1μgDNA使(shi)用(yong)(yong)3.0μLEndoFectinTM試(shi)(shi)劑都能(neng)獲得較高(gao)轉(zhuan)(zhuan)(zhuan)(zhuan)染效率(lv)。使(shi)用(yong)(yong)者也可嘗試(shi)(shi)每(mei)1μgDNA使(shi)用(yong)(yong)1~4μL體(ti)積線性(xing)PEI轉(zhuan)(zhuan)(zhuan)(zhuan)染試(shi)(shi)劑進行優(you)化。?如(ru)(ru)想申請(qing)PEI試(shi)(shi)用(yong)(yong)裝(zhuang)，請(qing)關注(zhu)我們的(de)(de)公(gong)眾號：Mine-bio，回復(fu)關鍵詞“PEI申請(qing)”，即可參加！?哪個品牌的(de)(de)PEI轉(zhuan)(zhuan)(zhuan)(zhuan)染試(shi)(shi)劑性(xing)價比較高(gao)？

PEIMAX是(shi)(shi)一(yi)款高(gao)(gao)(gao)度濃縮的(de)粉劑(ji)，大(da)量科學(xue)家選(xuan)擇PEIMAX，在內部自行(xing)制備低成本(ben)高(gao)(gao)(gao)效益(yi)的(de)PEI轉染(ran)試(shi)(shi)(shi)劑(ji)。多(duo)年以來，經過眾(zhong)多(duo)業內人士評審的(de)公(gong)開(kai)研(yan)究(jiu)和(he)出(chu)版文(wen)獻表(biao)明，在HEK293、CHO和(he)Sf9系統中可(ke)獲得很(hen)高(gao)(gao)(gao)的(de)轉染(ran)效率，在重組抗體和(he)病毒載(zai)體生(sheng)產中穩定性高(gao)(gao)(gao)，可(ke)靠性強。PEIMAX是(shi)(shi)一(yi)種(zhong)非GMP等(deng)級PEI轉染(ran)試(shi)(shi)(shi)劑(ji)，適用(yong)(yong)(yong)(yong)于(yu)基礎科學(xue)研(yan)究(jiu)及(ji)藥物研(yan)發(fa)早期(qi)階(jie)段。Transporter5轉染(ran)試(shi)(shi)(shi)劑(ji)是(shi)(shi)一(yi)種(zhong)即(ji)用(yong)(yong)(yong)(yong)型(xing)線(xian)性聚乙(yi)烯亞胺轉染(ran)試(shi)(shi)(shi)劑(ji)（PEI）衍生(sheng)物。由PEIMAX配置而成的(de)即(ji)用(yong)(yong)(yong)(yong)型(xing)無菌試(shi)(shi)(shi)劑(ji)，采用(yong)(yong)(yong)(yong)0.1μmPES無菌過濾器(qi)，完全消(xiao)除支(zhi)原體污(wu)染(ran)風險(xian)。適用(yong)(yong)(yong)(yong)于(yu)工藝開(kai)發(fa)、臨床前和(he)早期(qi)臨床研(yan)究(jiu)，可(ke)無縫過渡(du)到MAXgene用(yong)(yong)(yong)(yong)于(yu)cGMP生(sheng)產。如(ru)想(xiang)申(shen)(shen)請PEI試(shi)(shi)(shi)用(yong)(yong)(yong)(yong)裝，請關注我們的(de)公(gong)眾(zhong)號：Mine-bio，回(hui)復關鍵詞(ci)“PEI申(shen)(shen)請”，即(ji)可(ke)參(can)加(jia)！轉染(ran)效率好(hao)的(de)PEI轉染(ran)試(shi)(shi)(shi)劑(ji)是(shi)(shi)哪個品牌？天(tian)津高(gao)(gao)(gao)實用(yong)(yong)(yong)(yong)性PEI轉染(ran)試(shi)(shi)(shi)劑(ji)試(shi)(shi)(shi)用(yong)(yong)(yong)(yong)裝

MAXgene GMP是一種cGMP級PEI轉染(ran)試劑。申(shen)請PEI轉染(ran)試劑試用裝轉染(ran)步驟

接種細(xi)(xi)胞(bao)：轉(zhuan)(zhuan)(zhuan)(zhuan)(zhuan)染(ran)(ran)前(qian)，用膠(jiao)原(yuan)酶消(xiao)化細(xi)(xi)胞(bao)并計數（不建(jian)議用胰酶）。調整細(xi)(xi)胞(bao)濃度，將(jiang)細(xi)(xi)胞(bao)鋪入細(xi)(xi)胞(bao)培(pei)(pei)(pei)養(yang)的器皿(min)，總體(ti)(ti)積如(ru)表(biao)1所示(shi)，每(mei)個(ge)孔(kong)置入的細(xi)(xi)胞(bao)量應能使轉(zhuan)(zhuan)(zhuan)(zhuan)(zhuan)染(ran)(ran)時細(xi)(xi)胞(bao)匯合(he)(he)度達到70~80%。準備DNA-PEI復(fu)(fu)合(he)(he)物：DNA、PEI試(shi)(shi)(shi)(shi)劑(ji)和稀釋劑(ji)在進行以(yi)下(xia)步(bu)驟(zou)(zou)前(qian)需(xu)先(xian)使其升(sheng)至(zhi)(zhi)室溫。依(yi)據(ju)表(biao)1所示(shi)，用Opti-MEMITM（Invitrogen）或其他適(shi)合(he)(he)的無(wu)蛋白培(pei)(pei)(pei)養(yang)基(ji)(ji)稀釋適(shi)量DNA。用同(tong)樣的培(pei)(pei)(pei)養(yang)基(ji)(ji)稀釋PEI試(shi)(shi)(shi)(shi)劑(ji)。每(mei)1μgDNA需(xu)用2-5μL線(xian)性PEI轉(zhuan)(zhuan)(zhuan)(zhuan)(zhuan)染(ran)(ran)試(shi)(shi)(shi)(shi)劑(ji)。一邊輕(qing)(qing)輕(qing)(qing)渦旋(xuan)裝(zhuang)有(you)DNA溶液的試(shi)(shi)(shi)(shi)管(guan)(guan)，一邊將(jiang)稀釋的線(xian)性PEI轉(zhuan)(zhuan)(zhuan)(zhuan)(zhuan)染(ran)(ran)試(shi)(shi)(shi)(shi)劑(ji)滴(di)加(jia)至(zhi)(zhi)試(shi)(shi)(shi)(shi)管(guan)(guan)中（注意：請(qing)(qing)勿顛(dian)倒添加(jia)順序）。充(chong)分混勻后(hou)，室溫靜(jing)置10~25min以(yi)形成DNA-PEI復(fu)(fu)合(he)(he)物。當(dang)溶液體(ti)(ti)積較大時，請(qing)(qing)用圓底聚丙烯管(guan)(guan)，例如(ru)Falcon5mL/14mL離心管(guan)(guan)。轉(zhuan)(zhuan)(zhuan)(zhuan)(zhuan)染(ran)(ran)細(xi)(xi)胞(bao)：直接向每(mei)個(ge)孔(kong)中加(jia)入DNA-PEI復(fu)(fu)合(he)(he)物并輕(qing)(qing)輕(qing)(qing)渦旋(xuan)培(pei)(pei)(pei)養(yang)板/培(pei)(pei)(pei)養(yang)皿(min)。在無(wu)血(xue)清(qing)條件下(xia)轉(zhuan)(zhuan)(zhuan)(zhuan)(zhuan)染(ran)(ran)時，去除生長培(pei)(pei)(pei)養(yang)基(ji)(ji)，替換成無(wu)血(xue)清(qing)培(pei)(pei)(pei)養(yang)基(ji)(ji)，然后(hou)滴(di)DNA-PEI復(fu)(fu)合(he)(he)物。轉(zhuan)(zhuan)(zhuan)(zhuan)(zhuan)染(ran)(ran)3h后(hou)，添加(jia)?體(ti)(ti)積的包含30%血(xue)清(qing)的生長培(pei)(pei)(pei)養(yang)基(ji)(ji)。孵育(yu)細(xi)(xi)胞(bao)和分析結果：在CO2培(pei)(pei)(pei)養(yang)箱中37℃下(xia)孵育(yu)細(xi)(xi)胞(bao)至(zhi)(zhi)可以(yi)分析檢測。轉(zhuan)(zhuan)(zhuan)(zhuan)(zhuan)染(ran)(ran)后(hou)快7h即可檢測到轉(zhuan)(zhuan)(zhuan)(zhuan)(zhuan)入基(ji)(ji)因的表(biao)達。請(qing)(qing)自行確定(ding)檢測時間(jian)。更多關于(yu)Polysciences PEI相(xiang)關內容歡迎咨詢上(shang)海曼博生物！近期還可申(shen)請(qing)(qing)PEI轉(zhuan)(zhuan)(zhuan)(zhuan)(zhuan)染(ran)(ran)試(shi)(shi)(shi)(shi)劑(ji)試(shi)(shi)(shi)(shi)用裝(zhuang)！申(shen)請(qing)(qing)PEI轉(zhuan)(zhuan)(zhuan)(zhuan)(zhuan)染(ran)(ran)試(shi)(shi)(shi)(shi)劑(ji)試(shi)(shi)(shi)(shi)用裝(zhuang)轉(zhuan)(zhuan)(zhuan)(zhuan)(zhuan)染(ran)(ran)步(bu)驟(zou)(zou)

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