Or do the new school cloning, SLIC (Sequence & Ligation Independent Cloning) and "subclone" into any position in a vector. This method uses a PCR product and vector, the PCR primers have 20-40bp overlap with a region in the vector. Mix cut and purified vector with PCR product. Digest with T4 polymerase, quench, and transform. When I do the PCR in the morning I have clonies the next day.
REF: Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC Mamie Z Li & Stephen J Elledge Nat Meth V4(3) pp 251 Mark ------------------------------------------------------------------------------ Mark Collins Columbia University Biochemistry & Molecular Biophysics Black Building 259/201 Office/Lab 212 305 1951 (work) [EMAIL PROTECTED]
