Protocol 35540: Probe Assay - Ablim2<em1(IMPC)J>
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.

 

The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX). To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility. Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.

Expected Results

>chr5:35841020+35841129 110bp CAACCACATCCCAGAAGTCC GCAGGGCTGAGTCATAGCA

Mutant=  103 bp

Wild Type = 110 bp

Sequence

Wt Sequence (deletions in lower case; bp changes in brackets with wt first and insertions with carrots ^g^ (g insertion):

 TAGGGAGGATGCAGATGTGGTCCTTCAACCACATCCCAGAAGTCCTAGCACCaaggaggggtcagatggactggaaggctgggaatccaatgggcccatactagggccaagggtcttgctatgactcagccctgcctcactgtccagatgtgctctgtttatgcttgaagagcctgttagggttatgttgtttctgcccctgagatctagagggcaaatacagtgggcttggtgaagggcaagccgtgctcctgacccaggttttctcacactgtcttcctaccagggggatcaggatgatcggtcctacaagcagtgcaggacctccagccccagctctgccggctcagtcagcctcggacactacaccccaacctcacggtcaccccagcactacagtcgtccaggtattcacccccaccccactcaccttgacccccgtgaggttgtacttctccatgagtttcctgatacaaagtagcttctttattcattgagaaaagtggaaagggagggtggggggtggtgccggtggggccatggaatgtctggccacttagacatatgggaaagactgcctcaagcctgggagcaggcaaccataAGGCAGCTAGTCACCCTGGGGCTGGAGGAGGTGTCTGAATCCACAGTGCCCATCCCGAGACACACACAGCCCCAAGCTCCACACAGAACAACCAGAGTGGGCTTCTGGGGGCAGTTACAGAGCTGTCAGATGTGAAATTACACACGAGGTAGGAGAGCAAAGCAAGCCACAGTCTGTTGGGAGTCCAT

This allele was generated at The Jackson Laboratory by electroporating Cas9 protein and 2 guide sequences GAAGTCCTAGCACCAAGGAG and CCTGGGAGCAGGCAACCATA, which resulted in a 552 bp deletion beginning at Chromosome 5 position 35,841,047 bp and ending after 35,841,598 bp (GRCm38/mm10). This mutation deletes ENSMUSE00000185461 (exon 11) and 431 bp of flanking intronic sequence including the splice acceptor and donor and is predicted to cause a change of amino acid sequence after residue 350 and early truncation 37 amino acids later

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46327 CAA CCA CAT CCC AGA AGT CC Common A
46328 GCA GGG CTG AGT CAT AGC A Wild type Reverse A
46329 CTT GGG GCT GTG TGT GTC T Mutant Reverse A
46330 Fluorophore-1 CAG ATG GAC TGG AAG GCT G Quencher-1 WT Probe
46331 Fluorophore-2 CAC CAG GCA GCT AGT CAC C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46327 0.40 uM
46328 0.40 uM
46329 0.40 uM
Wt Probe 0.15 uM
Mutant Probe 0.15 uM
DNA

Cycling

Step Temp °C Time Note
1 95.0 --
2 95.0 --
3 60.0 --
4 -- repeat steps 2-3 for 40 cycles
5 40.0 -- Forever
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

This is the only strain that uses this protocol.