Protocol 33965: Probe Assay - Srsf5<em1(IMPC)J>Alternate1
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

>chr12:80947685+80947809 125bp GCTGAGAACCCAGAAACTTGA CAGGTGGAGCATTTCTGAAG

Mut= 126 bp

Wt= 125 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 GTGAATTGTTTTCCCAGAAAGATGTTAGAAGTGGCCAAGAAGCAGTGGCTTCTGTTAAGAAGTTAAAGTAACTGCTGGCTGGAGTTGATTCTCAGTCATCCCTAGCTTAGGGTTATAGTATCTGAGTGTAAGGTATATCTTAACACTTCCATGATTTTTTTTTTAAATttattttttatttttttggtttttcgagacagggcttctctgtatagctctggctgtcctggaactcactttgtagaccaggctggcctcgaactcaggaatctgtctgcctcccgagtgctgggattaaattaaaggcgtgcgccaccacgctcggcttccatgatcttaatgtgctgtagaatgcatgcacgtccaaaagctctactcttactgctacattttaggaatttgaggacccaagggatgcagatgatgctgtttatgaacttgatgggaaggaactttgcagtgaaaggtaagcatgattggtttataagtagttctcccagggaagtagagcagagtagacatttgcatatgtaagtgctctcatgtgtagtgtgtgaatgctttttttacctctttgtagggtgacgattgaacatgcccgggctcggtctcgaggtggaagaggtagaggacgatactccgaccgttttagcagtcgcagacctcgaaatgatagacggtatgtggtttgggtttggctgttacggttgggttagggtccgttagcattaaatggttctcagactagttaagtctagttagtccacatgctgagaacccagaaacttgatggaatcactatcttggcttaaacttgtcacatagcatttgacattgtgacataacatctgacactgaatgcTTATTTTTCTTTCTTCAGAAATGCTCCACCTGTAAGAACAGAAAATCGACTTATAGTTGAGAATTTATCCTCAAGAGTCAGCTGGCAGGTTTGTTGAAATACAGTTTTGAGATATTTTTTAATGTGGCATTTAAAAGTGTTAATGGGTAGTTAAT

This mutation is a 694 bp deletion beginning at Chromosome 12 position 80,947,084 bp and ending after 80,947,777 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
41158 GCT GAG AAC CCA GAA ACT TGA Wild type Forward A
41159 CAG GTG GAG CAT TTC TGA AG Common A
41160 CTG GCT GGA GTT GAT TCT CA Mutant Forward A
41161 Fluorophore-1 TGG CTT AAA CTT GTC ACA TAG CA Quencher-1 WT Probe
42553 Fluorophore-2 CAT CCC TAG CTT AGG GTT ATA GTA TCT G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
41158 0.40 uM
41159 0.40 uM
41160 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.