Protocol 35855: Probe Assay - Syne2<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

>chr12:75888482+75888621 140bp GTGGGCTTGGCTGATTCTG ACACAGTGAGACCCCCATTC

Mut= 135 bp

Wt= 140 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TGCCCTGTGCCTGCCTATTCCCTGTTTCTAAATGTTCCTTTGTTCTCAGTAGGTTGTAATCGGAGGGTCTGCATTGGAGGGTCTGCATTCCAGTCTGCCTGTTCTCTAAAGGTGTTTGATTAGGGTGTTAACCTGGTTCTTCTCAGTTTAATCCAGcagtgctggtggtgcccagtgggatctttagggaatgaatggaagccattcctgggtcaggtgcaagagacaccccggtccagacctaaggaaagccaaattgtagtcccaagtgtcaaatactgaactcagtcgtgtttagagctcatttcacccaaagtcctttgccagtgctaaagagtagttgccgagtagcatgtgctagcgtgtcagttaataactgcgctttccacagattgagaagctcgctcagactctgtcttgcgattacaatcaaccttcaccggaggttgtgagcgtggctgcctcatctcctacctcaagtcctcccactaagaagtgctccaaggctcaagctcaggcaagatggcagtggtctgccaagaaggctcttctgcagtgggctcaggagcagtgtgccaggtgagtagtttgctgtgccagatgagacagctgtgtccttccatccacacaacctcatggacttctggctctgacagtcgggctgatagtacactcccagcaacaccgtttcttagtgaatcttccagaagtggatcttggtgggcttggctgattctgctttgagaggtggcagctttaccctggttgctggctttccattgtttattgctgtgagCAGATAAAGAATATTTAAGTTTTCTAACTCCCCTTCCCTTTTTGAATGGGGGTCTCACTGTGTGGCTCTGGCTCTCCACAAACTCACACCCACCTACCTCTGCTTTATATATATGCTGGGAATAAAGGCACACACCAGCATGCCTGGCATTAAATATCTTTTTAAGGTACAGTTCTCCCATGC

This mutation is a 640 bp deletion beginning at Chromosome 12 position 75,887,919 bp and ending after 75,888,558 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
47173 GTG GGC TTG GCT GAT TCT G Wild type Forward A
47174 ACA CAG TGA GAC CCC CAT TC Common A
47175 GCA TTC CAG TCT GCC TGT TC Mutant Forward A
47176 Fluorophore-1 CTG GTT GCT GGC TTT CCA Quencher-1 WT Probe
47177 Fluorophore-2 AAG GTG TTT GAT TAG GGT GTT AAC C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
47173 0.40 uM
47174 0.40 uM
47175 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.