Protocol 36310: Probe Assay - Bltp3b<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

>chr10:89772129+89772239 111bp TTCTTTGCGTGGTGACTGAC AGAGAGAAGAGACAATGCTATGC

Mut= 119 bp

Wt= 111 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case):

 ACAAGTGGTGCTTCTAGAGTTAAGTCAGAGTTTTAAAAAGTTACCCAGAGAATATCAAAATCAACTTCTAAATGGGATTGTGTAATTTCCTTCCTCAGAAAAGTTAAAAACACTATACGTCAGAATTTATTTCTGAAGTGTTTTGGGTTGGTTTCTAAGGCTGGGAGTAGCAGGTGATCCTCAGAGGAGTGGTATGACCTCTGACCCTTGTTCTTTGCGTGGTGACTGACATCTGTGCCATGTGAGCTGCTGTTAGagggtcctctcaatttccctacctttgtgtgcttggttaatagcatagcattgtctcttctctctgatagggtctacagtgaacaaaatgtggtagggactgggcttgcattgtgcctggaaagcagtggtggatacacagttttcatccatttgagggagtcaggaccagacttcagctgtttacaggaatgatgtacacatcggggcccccaaactgtagactcactattagagagttgtggcctggagagcagagaacagcagtgacagacactggacatgggttaaaataaactcatctctgtggtggagtttagtctgctgatgtgtttctagaactctgtatttgctaggttgtctgtagggtgcattagctgacactaaagtaatgtaagtgtggtctctttcagattcactaaaaacttatctcctgacaaaataaacctcagcacccttaaaggagagggggagctgaagaacctggagctggacgaggaggtgctgcagaacatgctggacctgcccacctggctggccatcagcaaggtgttctgcaataaagcctccatcagggtaagcaaggcctggctgtctgcggaggctgcctgctctcacaatagatactcggcttcaatagatACTCGGCTTCAGAACTGCTTATAAAGAAACACTGTGTTCCTAGTATTTACACAGAAAATAGCTGCATGAATGGTTTTCATCCCTTATGAGTATAACTGAAGTAACATATTAACAACTTACATTTACATTTTGTTTAAAACATCTTTATTGATAGTTTAATAAAATTGATTTATTATAAATTAATTGCTTTACTTTGGCATATATTCAATGATAGCCCGCCCCAGGTTTTATTGTCAAATGAAATGTTTTAAATTTTTAATACTT

This mutation is a 641 bp deletion beginning at Chromosome 10 position 89,772,175 bp and ending after 89,772,815 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48394 TTC TTT GCG TGG TGA CTG AC Common A
48395 AGA GAG AAG AGA CAA TGC TAT GC Wild type Reverse A
48396 CCA TTC ATG CAG CTA TTT TCT GTG Mutant Reverse A
48398 Fluorophore-1 TAG ACT CGG CTT CAG AAC TGC T Quencher-1 MUT Probe
48405 Fluorophore-2 TCC TCT CAA TTT CCC TAC CTT TG Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48394 0.40 uM
48395 0.40 uM
48396 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.