Protocol 36650: Probe Assay - Specc1l<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

>chr10:75230953+75231047 95bp TTGGGGTTCACTCTCTGCT TTCGTACAGATAGACCAAAACACC

Mut = 102 bp

Wt = 95 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case):

 CTAGCCAGCACATGGTATAAGGTGTTCTGTCTCCTGAATGGGTTAGGACACACATTATAAATCTGTAAAGTATTAGCTTCTATAAGTGAACTGAAAGATTGCCAACATTTTTGTTTTGTTATTGAGACATCTGTCTTTCAGATACTTTTAtcatgtcgtggaaattttcagaaataatttctaaagagctgctttgtcgttttcttttcaacatgatgcctcttctattcttcccacaggtttggttgtaaatgcatcaccaagaggcagcccagaatgaagaaagcaaacaggagtgctggctcagtgcctaaggtgtctgggataagcaaaccgcaaacagtagagaaaagcaagcctgagaacagctcttcagcacccacaggagtcaagcctgtgaggcctggggcagcagcagccttgtcaaaggtattcacgtagctgtatttgaatatgcagtggattttaggcatgtatttaaatatgttgaatttattaaatgaatataatttggggttcactctctgctagtactaggaacacagtctttgaggagatctttcagcagGGTAACCTTAATTGGTGTTTTGGTCTATCTGTACGAATGAAACTTAACAGTAGGCTCTGTATAAAGAAAATAACCAAGTGCCTGCTTTATACAGGGAATCATGTACAGGTGAGCCAGAGGTAGAGTCTGCCTTCAGGGAGTTTGAGA

This mutation is a 408 bp deletion beginning at Chromosome 10 position 75,230,598 bp and ending after 75,231,005 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49320 TTC GTA CAG ATA GAC CAA AAC ACC Common A
49321 TTG GGG TTC ACT CTC TGC T Wild type Forward A
49322 GTG AAC TGA AAG ATT GCC AAC A Mutant Forward A
49323 Fluorophore-1 TAC TAG GAA CAC AGT CTT TGA GGA GAT CT Quencher-1 WT Probe
49324 Fluorophore-2 TGT TTT GTT ATT GAG ACA TCT GTC TTT CA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
49320 0.40 uM
49321 0.40 uM
49322 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.