Protocol 32476: Probe Assay - Relch<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

>chr1:105686820+105686979 160bp TTGCATGCTTCCTGTTTTCA TCCTTGGCTTTCCGTAGTTC

Mut= 126 bp

Wt= 160 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case and insertion with carrots ^ t ^ (t insertion):

 

TTAAATAAAAGTATTGTATTTTAATAGCTGTATTCCTTGTCAGACAGCTAGAACATATACCCCTAATTCTGCTAAAACTATGGAAAACGATCACCAGTTTGCTTGCAAATCAACTCTGCTGCATGCGTTCACAAGTTTC^ataaagg^TTGCATGCTTCCTGTTTTCAATACAAATTGTGTTTTTTTCATACCCATCTTTGCTTTTTATTTTTTTTATTTTTGTTTCCCTTttcttggttggatgcctgtgtgtgtttaatccaaatccctgtccagtcctggagtttgaactacggaaagccaaggagaccattcaggccctccgagccaacctaacaaaggctgcaggtggtgtacataaaactttttgagccttttcccacttttacaatgtagaagtagccggactatttttaagtgtaaaatagtaaaacattatctcattttatctcaccagaacatgaagttcctttacaggaacgaaaaaattataaatcaagtcctgaaattcaggtgggtgattgacaacaccagttatcttccatacaacccaagtgtcaaaggagcaaagctcacattttcccatctgtcttttaactttttttccaggagccaatcaaacctcttgaaaagagagctctcaacttcctagtcaatgagtttttgttaaagaataactacaagcttacatcaataaccttttcagatgaaaatgatgatcaggtacagttactgttttaagtaatgtctacttttaatctactgtttctctaacttgatttttaacacttatgcctgtaataagagtccatgtgggtagtttctaaaatcaaagcaggtactactcagtttagtattcaattggctcacaaaccttttctatttaaataaacctttttaaataaagacttatgtttctttccaggTAACTTTTTATAGCCTGATAGTAACTGTATAGCCCCTTCATGTTTGTATTCATTCATAATGTATGTTTTTCTAAGACTGAGTAATTACTTGCTTTTATTGATCATTGCAGACTTAGAAATTGAGTTTAACTATAAAAAAATTTCTTGAGTAGCACTGTATATAATTCAGATGTTCAGTAGCAATGGTTGGAGGTTTTTTT

 

This muation is a 696 bp deletion beginning at Chromosome 1 position 105,686,903 bp and ending after 105,687,598 bp (GRCm38/mm10). In addition, there is a single bp (T) insertion and 7 bp deletion (ATAAAGG) 83 bp before the 696 bp deletion that will not affect the results of the mutation. 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
39187 TTG CAT GCT TCC TGT TTT CA Common A
39188 TCC TTG GCT TTC CGT AGT TC Wild type Reverse A
39189 ACA TGA AGG GGC TAT ACA GTT ACT ATC Mutant Reverse A
39190 Fluorophore-1 AAT CCA AAT CCC TGT CCA GTC Quencher-1 WT Probe
39191 Fluorophore-2 TTG TTT CCC TTT AAC TTT TTA TAG CCT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
39187 0.40 uM
39188 0.40 uM
39189 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.