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?