I am doing cloning with pBD-GAL4 Cam. This vector has 4 unique restriction sites in the MCS (EcoRI, SalI, PstI, SrfI). My fragment also has SalI, PstI restriction sites, then I must do cloning with single RE digestion.
Anyone can give me advice, except using single RE digestion, preventing self ligation and ligation.
Thank in advance.
Hi there,
What about EcoRI and SrfI(which is contains SmaI/XmaI recognition sequence) in your insert then?
Whatever the strategy, you can dephosphorylate your linearized vector using (Antarctic, CIP or SAP) to prevent self-ligation.
If you want to use two sites then cut vector with EcoRI & PstI but cut your fragment with EcoRI and any of the available restriction enzyme (like BsiHKAI or Bsp1286I or NsiI or SbfI) that produces compatible cohesive end of PstI.
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