03 January 2020 2 1K Report

Hello everyone,

I am trying to insert 3 different fragments into a vector (size aprox. 8500bp)

Size of each fragment is around 248bp (fragment A), 705 bp (fragment B) and 1000 bp (fragment C).

I found out that multiple fragment cloning is very difficult if the classical cloning method is used.

But I was wondering if I can clone this by classical cloning method using T4 ligase.

The vector was digested with SalI and XhoI.

Fragment A was digested with SalI and EcoRI

Fragment B was digested with EcoRI and StuI (StuI is blunt end)

Fragment C was digested with StuI (blunt) and XhoI

I tried to ligate all the fragments at once with 1:12:8:9 molar ratio according to other's experiences but I have not gotten any positive clones yet.

Does anyone has any experience with multiple fragment cloning with T4 ligases?

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