Protocol 36064: Probe Assay - Avl9<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

>chr6:56723728+56723860 133bp GGTGAGACATGCACAAGAGA ATTGAAGGCACACCCATCAG

Mut= 118 bp

Wt= 133 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case; and insertions with carrots ^g^ (T insertion):

 TTCAAATCCCAGCAACCACATGGTGGCTCACAACCATCCATAATGAGATCTCATGCCCTTTTCTGGAGTGTCTGAAGACAGCTACAGTGTACTTACATATGATAAATAAAAATAAACCTAAAAAAAAAGAAAGAAAAAATTGTGAGTGTTCATGTAGAAAGGAGGTTAACTGTTTTGAAATCAATTAGCAAA^ctcatctaatttttaagtacaagttccaattgtagtaattttaccagcaggaaacaagaattctaaaaactgccaacataaggtaggcagtctacaatttatggatgaattgtgtttccagagttgctatttaaagcctggaccacattttcccaaagaaactgtgttacagacaccgtgaacccaattcttcctgacacggtcttaatttcacttacagaattcttgagtatgtttagcacctaaactctatttaaacatactcaactctatttaaactttgagttacaactagaccctttaaatcagctgggaatagtctataaaccttcccatctttctccacttctgcttttgagtttgttgataatcttcaatagcatcattcagggtttaagcacatgacagttcatcagtgtaagaccctttctttgatcactgtttggctaacattctttttcattttaggttgaattctcttacccaccactaattccaggagatggacatgatagccacactttacctgaagaatggaagtacttgcctttccttgccttaccagatggtgcacataactatcaggaaggtatgtcattaaatcgtagaccaattactaagtgcaattttggaaattttctcatagcggtcgtgaatactttggataaatgaaagcctttggacctgttgtgattacttataaaatgtgtatgtgttggaggtgagacatgcacaagagaagcttcagagagtactgggcctatttccttggttagtaaccctcgg^CTCCCTCCTGCCCAGAGCTCTTAGGTATAGCCAGAGCAGGGCTGCTGCTGATGGGTGTGCCTTCAATTATATTATGCTTGAGACTGAACTCAGTGGCAGAAATGAAGATTTGCCTGCATTAGCTGTACATGGTCTGGAAAGCTCTGGTTTCAGACTTACTCAACCATCATCCTCTAATGCCCACCTGGATACAGTGG

This mutation is a 786 bp deletion beginning at Chromosome 6 position 56,723,008 bp and ending after 56,723,793 bp (GRCm38/mm10). In addition, there is a single bp (T) insertion at the deletion site.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
47753 GGT GAG ACA TGC ACA AGA GA Wild type Forward A
47754 ATT GAA GGC ACA CCC ATC AG Common A
47755 TGA GTG TTC ATG TAG AAA GGA GGT Mutant Forward A
47756 Fluorophore-1 ACT GGG CCT ATT TCC TTG GTT A Quencher-1 WT Probe
47757 Fluorophore-2 CAA ATC TCC CTC CTG CCC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
47753 0.40 uM
47754 0.40 uM
47755 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.