Protocol 31719: Probe Assay - Ribc2<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

>chr15:85132604+85132707 104bp GGTGGGATCTACACCAGCAC TTCCTATATACACACACTCCCAACA

Mutant=  120 bp

Wild Type = 104 bp

Sequence

Wt Sequence: CTTGCAGTAGTGAAATAGGCAGACTTTAAAACACAGAAAATGTGGTGGGATCTACACCAGCACTGACTGGAGCCTGGAGGGGAAATCAgagagggcttcacagagtagtggtacttaaacaatgttgggagtgtgtgtatataggaaatgaaggaagctctggtgacagctgacagcgcaggcagaagacccagccataagcaaaggcttccacgtatgggaagggaacctttaatgctgtggccagatgagagggttcgtggcctactgaataatgctagctggctctaacccctcttcccttgtccattcctagggagacacacaagcctgggattttcaagtccgtgaccagaagataaaagaaataactgacaaagctaggcatgaagcctttggtgagcacttctaaaatgcccagctatttcttacgGCCTCTAAAATATCAGCTATCTCTTAGGGCCTCATGTCTTCCTCTTACAGCTGAGAAGAACAGACACACACACACACACACACACACACACACACACACACACGT

 

Deleted Sequence: gagagggcttcacagagtagtggtacttaaacaatgttgggagtgtgtgtatataggaaatgaaggaagctctggtgacagctgacagcgcaggcagaagacccagccataagcaaaggcttccacgtatgggaagggaacctttaatgctgtggccagatgagagggttcgtggcctactgaataatgctagctggctctaacccctcttcccttgtccattcctagggagacacacaagcctgggattttcaagtccgtgaccagaagataaaagaaataactgacaaagctaggcatgaagcctttggtgagcacttctaaaatgcccagctatttcttacg

345 bp deletion beginning at Chromosome 15 positive strand position 85,132,649 bp and ending after 85,132,993 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37483 GGT GGG ATC TAC ACC AGC AC Common A
37484 TTC CTA TAT ACA CAC ACT CCC AAC A Wild type Reverse A
37485 TGT GTG TGT GTC TGT TCT TCT CA Mutant Reverse A
37486 Fluorophore-1 AGG GCC TCA TGT CTT CCT CT Quencher-1 MUT Probe
37487 Fluorophore-2 AGA GGG CTT CAC AGA GTA GTG GT Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37483 0.40 uM
37484 0.40 uM
37485 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.