小中大紧急请教:珍贵样品P不出
紧急请教:珍贵样品P不出[转载]
珍贵样品模板:是有限的别人提供的少量pcr产物(96孔板那种)长约4kb
pcr system:roche expand high fidelity
老板预先将样品稀释了n倍,给我,让我扩出来(预先将原样品1ul跑胶,无带)
我做了nested pcr
first round 50ul
standerd roche expand high fidelity pcr system
template dna 1ul
primer 260nM
Mg++ 1.5uM
95 4'
94 1'
45/50 1.5'
68 5'
40cycles
结果 jin仅见很亮的引物二聚体带
second round
standerd roche expand high fidelity pcr system
template dna 5ul the products of first round
primer 260nM
Mg++ 1.5uM
95 4'
94 1'
48/52 1.5'
68 3'
40cycles
结果:成片长长的很亮的拖带
请高手指教
样品 仅剩5ul