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.
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
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: