This is the third step in the KOMP-CSD/EUCOMM production centre QC pipeline. For conditional constructs , this test (the 5’ Screen) is only performed If no long range PCR (LRPCR) bands are generated in the 3’ Screen.

LRPCR is performed between a universal primer in the 5’ arm of the targeting cassette and two different gene specific primers in the region upstream of the construct region.

The subsequent LRPCR amplicon is sequenced ( R1R primer [5’-CGTGGTATCGTTATGCGCCT-3’] ) to verify the gene.

Possible Outcomes

This data originates from the HTGT database. In HTGT this QC test is called “5’ arm QC” and can be reported with the following results:

• na = LRPCR not attempted
• nd = no LRPCR or sequencing reads detected
• fail = LRPCR and/or sequencing read detected, but does not meet pass criteria
• pass = LRPCR 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 GF LR-PCR band + valid R1R sequence read
• 2 x GF LR-PCR band

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