Protocol 36867: Probe Assay - 1700030J22Rik<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

>chr8:116972090-116972187 98bp GCTTGAGGGTGACCTGCTAC TCCCCCTTTTCACTTTCTTC

Mut = 101 bp

Wt = 98 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case):

 GCCTGGCGTAGTTGGCTTGAGGGTGACCTGCTACTTTCTCTGCAGGTCCATggctggggaaggacctcaccaactgcctgcatctggtcaaagaagaaagtgaaaagggggagggccagagaaggcaccaatggtggcaacgactgcttgttctgtatgagtctttcccaagggagtctggagccccggtccctcttggaggttgggaaactggaggcgggtgctgaggccgaggtgagcacccagaagagctggagctctgagaagaactggagcggcctctcccagggccccggcactgcttccagagagcagtctaacaaactctgcattcccaccgacgtgcatggagaaaaaaaaagcctgcaactcaaggagtttatctggtgcatggaggagtggcctatgcccgagacagttagcagcaaggccggcaggaaccccagcggaagccccgagcaagggctctccacccctgactcccttgcagccaaggctcttgtggttcttcccccgctgaaaagctctccccacaacttggacgtcctgagtaagaaaagcaggaatattttctggcagccggaagaaaaggtgctgagggtggaaaaagacgattgtatggcttgtgcagatggactgaaaggagtagacgggaaaggtgaaaagagacactttgagctggccagccacgtgaaggtcaccaacgtactgcctttcccacccactgcggcccagacccacctgctgtcagccgagtcccaaaggtgctgcctgcactggtccctcctgccccagaaaagcactgtgttcccacctaaccccagcgacatccactaccttgccaccctgcaggttttggggcaacagggaaagcaaagctgcagaactaggctcaaaaccaaggacacaaaacctcccaggaccactgccaagcatattatcacagaggccaagcagcaaaacaggccccacgtgctAGAAAGCAAAGTCTTCCCTAAACCTCTCTTGCCA

This mutation is a 926 bp deletion beginning at Chromosome 8 position 116,971,225 bp and ending after 116,972,150 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49930 GCT TGA GGG TGA CCT GCT AC Common A
49931 TCC CCC TTT TCA CTT TCT TC Wild type Reverse A
49932 GGG GAT GAC AAC TCT GCT CA Mutant Reverse A
49933 Fluorophore-1 TGG GGA AGG ACC TCA CCA AC Quencher-1 WT Probe
49934 Fluorophore-2 TCT GCA GGT CCA TAG AAA GCA AAG T Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
49930 0.40 uM
49931 0.40 uM
49932 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.