Protocol 37517: Probe Assay - Insyn2b<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

>chr11:34401957+34402046 90bp TCCATGGCCCAGCAAAGT GTGGTGCGGTTGTTTCACA

Mut = 99 bp

Wt = 90 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

CCAGGACAAGCGTGCGGTTCTTCTCAGTGAGGCTCCATGGCCCAGCAAAGTATgaaagtgaggcctgtgctcctaaagcgaagcagccttgagtctgtggagcttgtgaaacaaccgcaccaccgcaggagcaaatcgcagcaagtgcgattcaaggaagatggcaacattaagaatccaactggcgtgaccgaggtcaacacccaaacccccgaagacccaggtgtaatggggaaggcacaggcctccaggcaccatcatcctaccacctactcgctgtctttccccaggtcccacaaggcaggaggattccgcagcatctccatccagacctcccccagtctcaggaagcatttccccgttttcaaaaggaagaaactcacgactagcaagtccctggtggaaatgccaacagcatccccaagtgccatccaggtcaacggcaacctatctgaacaggatattgtgtcctctgaccttgccttcctgaggctggctcagcatcttgaggatgggcctcgcaggctcaaaatcccccacccatttctccctaggatgcccaaggtgcaaagcaatgggcctgtcagcttctgcttagaatctgggacttggatgtcctcagagaaagccacagctgctattcaggttccagatgatatttgtcacagtccaacctgggaagctagagagtcggctctcagcccagaaagctcagctgaagagagcaactctatacctgctttgatctccatgtgtccaggggatgggcaaagagtgatgacgtcagagctggaaagaatgcccccctgctcaaataccaatagcagtgccagtaatatgccgggcacagaaaaactcacacccgaattgcttttgcccaaagacaaccctgatgacaaagaccttggccttccatcatctcagtcaaagaagatgtgtgttccctcaccaccacggactcacagttcgcctgagccaggctcccgtagccagccagtccacctaggaagatcttctgattgcccagcctcaggtgacaatcaccaggacctggagtcactcagaagtagcagtgcatccaagtctgtccctgtgtgctgggaacatgtgacaaagctccccagccagtcagacaccccagagcttcagactggtgttggaagtgagcaactccctgcctcgattccaaggcaggagaacagagctcagagcagcagggagataggtgggagtaatcacagtcacctggcgcagggagagctctgtgacctccaaggccggctgcaatcagtagaggaatcgctgcactcaaaccaagagaaaattaaagtcctattgaatgtgattcaggatctggagaaagctcacgctctcactgaaGGGTAAGGTGAATTTCTTTATGTTGAAAAACCACTGAGCCGAGTTGTTTTCTGGGAAGGGAAAGAATGAGAAAGCAAGGTCTCCCACAAAAAGTTCTTGCTCTGTTGTTTACCTAGACGCTA

This mutation is a 1327 bp deletion beginning at Chromosome 11 position 34,401,977 bp and ending after 34,403,303 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
51552 TCC ATG GCC CAG CAA AGT Common A
51553 GTG GTG CGG TTG TTT CAC A Wild type Reverse A
51554 CCT TGC TTT CTC ATT CTT TCC Mutant Reverse A
51555 Fluorophore-1 CCT AAA GCG AAG CAG CCT TG Quencher-1 WT Probe
51556 Fluorophore-2 ACT GAG CCG AGT TGT TTT CTG G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
51552 0.40 uM
51553 0.40 uM
51554 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.