Protocol 34653: Probe Assay - Sh3rf3<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

>chr10:59071655+59071752 98bp AGGATAAAGCTAGGCCTCTACC GTTGGTGGCTCAAGGCATCT

Mut= 90 bp

Wt= 98 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 GAAACTGCTTCCACGATGGAACACAGTGTTTGAGGAAACCTAAATCTGTGTTCCCAGGCCATAGCCGTCTAAATTGGCTTACAGAACCTTTCATATAATTTTCATTTCCACACACTAATAATTAGTCCAGGTTCTAAGGATAAAGCTAGGCCTCTACCCGACAGGGTCTCCTCCCATgttgaccttgggatggtacttagaaggggcccttgaaagatgccttgagccaccaacctcaatgcaacaggatagcatatgtgattcctagccaaggaagtaaggcgtgaggactgggaagatggaggctttgacaccgagggaatggatacccgtggctgctgggggcctactgaaccacttgttccttaagtgagacctttcttctactcactcacgtatcctcttgtgtcttttcaggactcctcttccgctggacctgtccccacagccctcccacgagctgctgcagtggctggagaacagggcatgtctcccaaagtccagctgcccctcaacgtgtgagctgcccctcatgtctGTCGACATTCTCTGCACACTGGCATTTACACAAGTGGTTCCTAGTGGGTTTTTCAGTTGGTTACTAGCTGTTCTTTGTAGTCGCACTTGTGTGTCGATGCTTCCCTGAAGGGGAGTGTGAAAAGTCTCTTAACGCTGCTGTCCCTAGGATAATTAGGAGAAGTGAGGGAAGACTAATCACACTGGACAGCA

This mutation is a 361 bp deletion beginning at Chromosome 10 position 59,071,696 bp and ending after 59,072,056 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44157 AGG ATA AAG CTA GGC CTC TAC C Common A
44158 GTT GGT GGC TCA AGG CAT CT Wild type Reverse A
44159 ACC CAC TAG GAA CCA CTT GTG T Mutant Reverse A
44160 Fluorophore-1 ACT TAG AAG GGG CCC TTG AA Quencher-1 WT Probe
44161 Fluorophore-2 CCA TGT CGA CAT TCT CTG CA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44157 0.40 uM
44158 0.40 uM
44159 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.