定量PCR實驗故障排除

1、定量PCR實驗故障排除王爭強Hotline: 400 620 9660cnmcbsupport擴增曲線-v1.4 2009 Applied Biosystems. All Rights Reserved.擴增曲線-v2.0 2009 Applied Biosystems. All Rights Reserved.標準曲線-v1.4 2009 Applied Biosystems. All Rights Reserved.標準曲線-v2.0 2009 Applied Biosystems. All Rights Reserved.原始數(shù)據(jù)-v1.4 2009 Applied Biosystems

2、. All Rights Reserved.原始數(shù)據(jù)-v2.0 2009 Applied Biosystems. All Rights Reserved.原始信號-v1.4 2009 Applied Biosystems. All Rights Reserved.原始信號-v2.0 2009 Applied Biosystems. All Rights Reserved.融解曲線-v1.4 2009 Applied Biosystems. All Rights Reserved.融解曲線-v2.0 2009 Applied Biosystems. All Rights Reserved.常見問

3、題一：沒有標準曲線1 2009 Applied Biosystems. All Rights Reserved.常見問題一：沒有標準曲線 2009 Applied Biosystems. All Rights Reserved.常見問題二：沒有擴增曲線擴增曲線組分 2009 Applied Biosystems. All Rights Reserved.常見問題二：沒有擴增曲線原始數(shù)據(jù)只有1個循環(huán)光譜 2009 Applied Biosystems. All Rights Reserved.常見問題二：沒有擴增曲線原因1：PCR參數(shù)設(shè)置錯誤 數(shù)據(jù)收集步驟只有一個循環(huán)，只收集了一個數(shù)據(jù)點。 20

4、09 Applied Biosystems. All Rights Reserved.常見問題二：沒有擴增曲線原因2：硬盤休眠，數(shù)據(jù)采集中斷 2009 Applied Biosystems. All Rights Reserved.常見問題二：沒有擴增曲線原因2：硬盤休眠，數(shù)據(jù)采集中斷75007900 2009 Applied Biosystems. All Rights Reserved.常見問題三：基線不平滑 2009 Applied Biosystems. All Rights Reserved.常見問題三：基線下滑從原始數(shù)據(jù)找原因 1-9循環(huán)，ROX的信號下降，與試劑質(zhì)量有關(guān) 基線設(shè)定

5、4-13范圍內(nèi)，ROX信號值變化，F(xiàn)AM的信號值基本固定；FAM/ROX的校正后，基線不是水平的直線； 應該取ROX信號穩(wěn)定的10-20循環(huán)為合理的基線范圍。 2009 Applied Biosystems. All Rights Reserved.常見問題三：基線下滑修改基線范圍為10-20，重新分析 2009 Applied Biosystems. All Rights Reserved.常見問題三：基線下滑基線范圍取10-20循環(huán)的數(shù)據(jù)，重新分析后的圖譜； 2009 Applied Biosystems. All Rights Reserved.常見問題四：擴增曲線彎曲原因：模板濃度高，

6、基線的終點大于CT值通常是因為模板DNA濃度偏高，CT值 10000點，就不會打折Rn=弱的信號經(jīng)過校正后，點與點之間的差異被放大 2009 Applied Biosystems. All Rights Reserved.常見問題七：擴增曲線不光滑解決方法 使用高質(zhì)量的探針 補救方法：關(guān)閉ROX校正，重新分析數(shù)據(jù)關(guān)閉校正 2009 Applied Biosystems. All Rights Reserved.常見問題七：擴增曲線不光滑關(guān)閉ROX校正的效果 2009 Applied Biosystems. All Rights Reserved.常見問題八：重復性不佳 2009 Applied

7、 Biosystems. All Rights Reserved.常見問題八：重復性不佳 2009 Applied Biosystems. All Rights Reserved.常見問題八：重復性不佳原因：蓋子未蓋緊，溶液蒸發(fā) 2009 Applied Biosystems. All Rights Reserved.常見問題九：參比熒光 2009 Applied Biosystems. All Rights Reserved.常見問題九：參比熒光設(shè)置不當 2009 Applied Biosystems. All Rights Reserved.常見問題九：參比熒光設(shè)置不當 2009 Applied Biosystems. All Rights Reserved.常見問題九：參比熒光設(shè)置不當 2009 Applied Biosystems. All Rights Reserved.常見問題九：參比熒光設(shè)置不當 2009 Applied Biosystems. All Rights Reserved.