This is the second step in the KOMP-CSD/EUCOMM production centre QC pipeline. Long range PCR (LRPCR) is performed between a universal primer in the 3’ arm of the targeting cassette and two different genomic specific primers in the region downstream of the construct region.

The subsequent PCR amplicon is sequenced with a primer to the downstream loxP ( LR primer [5’-ACTGATGGCGAGCTCAGACC-3’] ) to verify the gene and loxP site for conditional constructs or is sequenced ( R2R primer [5’-TCTATAGTCGCAGTAGGCGG-3’] ) to verify the gene for deletion/insertion constructs.

Possible Outcomes

This data originates from the HTGT database. In HTGT this QC test is called “3’ arm QC” and can be reported with the following results:

• nd = no PCR or sequencing reads detected
• fail = PCR and/or sequencing read detected, but does not meet pass criteria
• pass = PCR and/or sequencing read meet pass criteria.

Pass Criteria

In order for a ES cell to be called a “pass” for this QC test it has to meet one of the following criteria:

• 1 x GR LRPCR band + valid LR sequence read (Conditional constructs)
• 2 x GR LRPCR band (Conditional and deletion/insertion constructs)
• 1 x GR LRPCR band + valid R2R sequence read (Deletion/insertion constructs)

This logic is explained in more detail in the following flow chart: