For the purpose of gene targeting vector construction, mouse
BAC genomic libraries have advantages over other types of genomic
libraries. Because the average size of insert for BAC clones is
around 150 kb, the chance is high for having all the necessary
pieces in one BAC clone. Another advantage is that many mouse
genomic BAC libraries are commercially available.
The purpose for BAC library screening is to identify the clone
ID of the clone containing the gene of interest so that we can
order it for targeting vector construction.
Because a unique and pure southern probe is critical for the
success of BAC library screening, a rigorous procedure is required
for making the probe.
A typical BAC library screening involves the following steps,
Design the probe primers within the gene of interest
PCR the probe using the matching genomic DNA as the template
Clone the PCR fragment into a vector
Sequence the vector insert to confirm that its sequence
matches that of the GOI
Label the probe with P32
Southern blot of the library filters
Interpret the result
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