Protocol 35785: Probe Assay - Specc1<em1(IMP)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

>chr11:62119483+62119590 108bp AAACCCACCCAGATTTCTGTA ACACAGGTCACCCCTGACG

Mut= 142 bp

Wt= 108 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 AGAAACCCTATTCAAAACAGATTTTGAGCATGTCACTGCAGACACCCCATCTCAGCCCCTGTCAGCCGCTAGCAACCCTTTTAAGAGCTCCAAGGGCTCCCCTACTGGGAACTCCCCAAACAATTCAAATGAGTTGTCCTTGCCGCTCTTACTGAGAAGatacctagaggaggaagaattggatgaagagtgacagccatagcatcatctatgccatatatggaagacatttgagtttcattataaaattttttttctttcttttttgtttcattttttctttcatcttttgtctttagggactattccaacaactaaacggacaggcattccagctccacgggagctctcagtaactatctccagagagaggtctgtgccacgtggtccctccagctcaaagaagttaggctccagcccaacttcttcctgcaatcccactcctaccaagcatctgaggaccacccctgcaaaacctaagcaagagcatgaaggtgcagagaaggctgtgctcgagtcccaggtacgagaacttctggcagaagccaaaaccaaagacagtgaaatcaacaggcttcgaagtgaactaaaaaaatgcaaagagaggtgggccctgagcactgaggatgccaatgcttcagacccaagtgcagagggaacagcatccccagaaagtgatgcacagcctttgataagaaccctggaggagaagaacaagacctttcagaaagagcttgctgacctagaggaagaaaacagggccttgaaggagaaactgacttaccttgagcagtcaccaaattcagaaggggcagcaagtcacactggtgacagcagctgcccaacatctataacccacgagtccagttttggaagcccagttggaaatgagttgtctagtgaaacagatgagtacagaagaaccacacatgggagtgccttgcggacatcagggtcttcaagcagtgatgtcaccaaagcctctctgtcaccggacgcctcagattttgagcatatcactgcagacaccccgtctcggcccctgtcagccactagcaacccttttaagagctccaagggctcccctactgggagctccccaaacaatgcaagtgagctgtccttggcatctctcacggagaagatacagaagatggaggagaatcagcacagcactgcagaagagctgcaggctactctgcaggaattatcagaccagcagcagatggtgcaggaactgacagccgagaacgagaagctagtggatgagaagaccattctggagacctccttccaccagcaccgagagagggcagaacagctgagtcaggaaaatgagaagctgatcaaccttttacaggagcgagtgaagaatgaggaacccagtgcccagggagggaaggtccttgagctggagcagaagtgcacggatattctcgagaagagccgctttgaaagagagaagctgctcaacattcagcagcagctgacctgtagcctacggaaggttgaggaagaaaaccaaggcgctatagacatgattaagcacctgaaggaagaaaatgagaagctgaatgggttcctagaacatgagcggtgtaataatagtgtgatggccaaaactctggaggaatgtcgagttaccttggaaggcttgaaaatggagaatgggtccttgaaggctcttttggaggctgataagcaaaaagccatagaggccagcagtactgtggggcagacagcagagaacttcgaggttcaggagatgctgaaagtggctcgagctgagaaagatcagttgcagctgtcttgcacagagctcagacaagaattgctaaaggcaaatggtgaaattaaacatgtttccagcctgctagctaaggtaagaagacagttctcaactagtatttgttgacatttctctttgagaccttaacattgtgaaccaaagagaatcaattattcaactgtatttgtgaatctaagccttctgatttggaaatctattagaatttaggcaggttagctctggggattccaaattttaaaacaaaacaaaacacaaaacaacaacaacaaaaaaaacccacccagatttctgtacagatagataaaaagattagtggaaatactgtaaattacatcagatgatatggtgtagTGATGAAGACCGTCAGGGGTGACCTGTGTCTTGGTGGTAGAATACTTTCATAGCCTCCTTGAGACTTTAGATTTGATCCCTAGCTCCACACAAAAATAGGATAAAATAAGATTTAGAACCATCCTGTATACATTGATGGACTTTTTGATACAT

This mutation is a 2002 bp deletion beginning at Chromosome 11 position 62,117,560 bp and ending after 62,119,561 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
47023 AAA CCC ACC CAG ATT TCT GTA Wild type Forward A
47024 ACA CAG GTC ACC CCT GAC G Common A
47027 CCA TCT CAG CCC CTG TCA Mutant Forward A
47028 Fluorophore-1 AGT GGA AAT ACT GTA AAT TAC ATC AGA TG Quencher-1 WT Probe
47029 Fluorophore-2 AGA GCT CCA AGG GCT CCC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
47023 0.40 uM
47024 0.40 uM
47027 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.