[求救] re-ligation的問題
大家好
就是，如果我有很多個dna片段，用限制酶有specific的overhang後
分組用T4 ligase作用overnight後，純化再接出全長
ex: ABCDEF個fragments，AB CD EF各自接好後，再AB+CD+EF接
想請問
在第二次ligation時會需要注意甚麼嗎？
當初限制酶切出了overhang會有可能再第一輪接完cleanup的過程中掉掉嗎？
第一輪的T4 ligase會需要inactivation嗎？如果沒有會影響religation的效率嗎？
歐然後想順便請問有人遇過接出大片段的DNA(~30kb)後跑膠與Marker不符的情形嗎？
我看paper有些人indicate的產物(明明應該比marker大)跑膠後卻比marker低
因為我自己也遇到了類似的情形，我的產物(~30kb)怎樣都比marker 25 kb的band低
就不知道是真沒接到某個片段(那次實驗室六個片段pool一起接的)
還是這是有可能的orz...
謝謝大家幫忙解惑
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