ES cell clone QC
Tony West
1. QC process and interpretation
2. Web based QC data navigation
European Conditional
Mouse Mutagenesis
Program
EUCOMM Informatics Training Day 1 December 2008 Hinxton, Cambridge
ES cell clone QC Pipeline
ES cell clones (8-96 clones) - (96 well plate format)
Isopropanol ppt clean-up of DNA
LRPCR + sequence verification
Mark for distribution
Recovery pipeline
EUCOMM Informatics Training Day 1 December 2008 Hinxton, Cambridge
5’ + 3’(loxP) gene specific PCR confirmation
Distribute
Cost effective LRPCR +
Sequence verification
• New automated primer design program
– ‘Primer Brain’
• New LRPCR kit
– ‘SequalPrep LRPCR kit’
• Joint development with Life Technologies
• One set of reaction conditions for all knockout designs
• New sequencing dilution buffer
– Sequencing reaction diluent (SRD)
• BigDye! used at 1/128th normal concentration
EUCOMM Informatics Training Day 1 December 2008 Hinxton, Cambridge
Primer Brain (PB)
• Parameters
– Length – 24mer to 30mer
– GC content – minimum of ten bases
– Min 64°C Tm
– C or G required at 3’
– Max 3 nucleotide run
• Process
– Critical region and 2kb blocks outside homology arms
– Sequence tiled at 1 base intervals for a stretch of 24 bases to 30 bases
– Repeat regions masked
– Sequences meeting specified parameters, screened for pairs
– Selected sequences blasted against the genome (<10 hits)
EUCOMM Informatics Training Day 1 December 2008 Hinxton, Cambridge
‘Primer Brain’/SequalPrep LRPCR kit validation
~1 kb
~5 kb
~5 kb
phase
Cassette
loxP
2 1
1 0
0 0
0 0
0
2
~2 kb
~2 kb
gf3/gf4
gr3/gr4
Ex3/Ex5
5’ LRPCR
3’ LRPCR
EUCOMM Informatics Training Day 1 December 2008 Hinxton, Cambridge
Using 2 selected primers for both 5’ and 3’ regions = >90% hit rate
3’ LRPCR + Sequence validation
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