Protocol 34480: Probe Assay - Insyn2a<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

>chr7:134918843-134918938 96bp ACATAAAGGCCGACCTCACC GAAGCACACATGCAAACGAG

Mut= 93 bp

Wt= 96 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TATCAAACGAAAACCAAGTGTATCCCACATGGAAAAAGTAAGCAGGCTTTAATATCGAGAAGCAGTTCACGCTGTGCCTGCCAGCAACTCCCCGCCTGTCTGATTTTCCTTAACATAAAGGCCGACCTCACCTGTTTAAAAATAATTAGGAAGCCAGCAGctgtgatttggctggacgtccattcggcctcgtttgcatgtgtgcttctgacccactgatgcctctgttgctgtctctgcatgcaggcaccatggtcagcaaggactctggcagatgcatactcacgacaccagagcgggaagtggagcccgccgcctgcctggccctggagatgagatatgccctcgaccctaaccggcagatcaaaaagcgaaacaaagctctgcaggtgcgttttaaggatatctgtgaggcccagaatgagcagagagacacacagctgtcttcgggacctctgggggagaagcgagaggccaaggctgtgtcctgcagggtggcctatcgtaagtatatgacagtgcctgcacgccgatccattcccaatgtcaccaagagcaccggtgtgcagacctcaccagatcttcgcaagtgttaccagacattcccgctggaccgcaagaaaggcagtctcaaaggcctgccggctgcagatgccttcaaaagccagaacaatgggtttctagcagattcaaaagagaagagcgaggcgggacccatggaggagcctcggccttgcagtgcaggaaggattcacaagaccaccgcattggttttccattccaatgagcacgtgaacgcactgggacagccttctggggtcaactgtgcagagctctgtaagtctccagatgtgcttggctacccagaggctgttttgcaaaactctcggcctccctctgaggagcccatccaccagctgcaggggagagctaagcttgacaggggcaccctggactcagaggagccggctccactcacccacggaagggtgtttaaaaccgaggtcgccactgtgtatccgcctgccatcagtaccagggcccctgagcctggcttgtcaaactctgctgctgccagccagtggtctctctgccctgcagatgaggagcagaggcgagccgcccacctcaatgggctacaggcatccacaggctctgctgtggcctgctcaaccccggtgcagtacctgtccccagaatgtagcgaacagcccctgcagaaccagcctagccccacagctgggataggggatgaagaacaccaacaaattgtgcctcacacagaggtggtagacctcaaggcccagctgcaggtgatggagaacttaatcagctctagccaggagaccatcaaagtgctgctgggagttattcaggagctggagaaaggagaagcccatcgggaagggtatgtatggccactgcatcttaccccctgtgggctcaccacttgcaggcccatcagcagccccgatgtcccagccctaaggagtctaatgccggtggtttttgtgtcagggctgcagtctgacaagggtcacagGTGTGTTGAAGGCCAGGCTTCCTGGCACACTCGCCTCTGATACTCATTCGTTGGTTAGCATTTACCCATCTGGCCTTGGGTTTCAATCTCTCCCTGTCTATTCAGGGAGCTGATGGGTCTTCCTTGTGATCTTTTCAGATTGATGTTTTCTA

This mutation is a 1383 bp deletion beginning at Chromosome 7 position 134,917,508 bp and ending after 134,918,890 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
43723 ACA TAA AGG CCG ACC TCA CC Common A
43724 GAA GCA CAC ATG CAA ACG AG Wild type Reverse A
43725 GAG TAT CAG AGG CGA GTG TGC Mutant Reverse A
43726 Fluorophore-1 ATT TGG CTG GAC GTC CAT T Quencher-1 WT Probe
43727 Fluorophore-2 CCA GCA GGT GTG TTG AAG G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
43723 0.40 uM
43724 0.40 uM
43725 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.