Protocol 32516: Probe Assay - Tspan9<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

>chr6:127967488-127967586 99bp TCTGCCCAGAGGAGTAACCT TCATGAGGTCTCTCTGAAGATCA

Mut= 121 bp

Wt= 99 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case):

 

ACCCATGGACTTGCTTTCCAGACTTCAGAAAACAGGGGTTACGTGACCCTTCTGTTCAGGGTAGTGGGAAACACCCACTGAGGTTTTGGCTTTGAGAGCCAGTGACCTGGGTTCTCCCCCGTAGACCAACCAGTCATACCCAGACCCAGCCTCCCTTCTGAGCATGGGGTCCTCTCTGCCCAGAGGAGTAACCTGACACACATTTGTGTAGTTTTCTGGTTTCCCCcttcgtctcactgaacactgaagttgatcttcagagagacctcatgaggtatatgaagctgcctcgtcccatttactgtaggggagaccgaggcatgcagtgacaagggggcatgtcataggctttgggtctgtgacagcgcctggctttttataatcattttgtatctcagagtcagtggtgagagacatgcatggggtggggtggtctgggagatgaatgtaagagctgtgctggtctaaccagtttcttctttctccagctctgtggctgtgggctgcttggagtgggcatctggctctctgtgtcccaaggcaacttcgccaccttctcccccagcttcccctcactgtccgcagccaacctcgtcatcgccatcggcaccatcgtcatggtgacgggcttcctgggctgcctgggggccatcaaggagaacaagtgtctgcttctcagcgtaagtcggccctcgcgttcccttcgttgctctctggggctccctagctgaagtccacagacattgcttctagagcgttctgtccccagcccttcctgtccccctctctctttgacacatgcctgggtttgcttaggagtgggtggtgcccacttccaggctcccagcctggaaaccctgcccatcccttatctcccctcccctgccccattctctgtcaccacccaccccggccatctccactcccctgggcccacgcttgcacttgtctttccagttcttcatcgtgctgttgatcatcctgctggcagagttgattctgatcatcctcttcttcgtctacatggacaaggtaaacctcgcagaggcaggcaagatggaaggtcgccatcttgacaccagggctgggacaggggaacgcctggctaacagatcctgcagactggtctgcaggaggcctgagggtcccaccctagccacttcccctccccgtctggaaacactgctccagcattcccagctggcccagtctgggttagggaggagggccagtggcctggtctcagctgacctgtggtgtttcccctcatctgccaggtgaacgagaatgccaagcaggacctgaaagaggggctgctgctctacaacacagagaacaacgtggggctcaagaacgcctggaacatcatccaggccgaggtgcggctccagtctgtgcatggcgtaggatggggcagccatagtgcacctgtgctgttgggagaaccacctgcactggacacttgcctagacctgaagggagatgctagcatgggatgctgccggggaggggtaaagaggtgggcgcacgaggcacatgatgcgtgtctcaggagtgggcgagcctaacgtggtgaatgacaggagtcaccagatcacgtttggctcaggtgacaggctctggtagtgacaggaatacaagtctctgttgcccgatgcacaccccacatagaccctgccaatcagcttgggagggcccgtactacaaaggaaggaggtcaaggcactggtgagcaagtattgggctgggctctttaactccacatctggtgctcagcccagagggacagcagcctttctggtgtaggaaagaagagtcactcactacctctgggggcccggctgcagctcaagaggggacaaggagacattcttttccctatccctgtgttcttgggtggggttgacgtatctggccttgtgccccacagatgcggtgttgtggggtcactgactacacagactggtatccggtgctcggggagaacacagttcctgacagatgctgcatggagaattcccagggctgtgggcgcaacagcaccacccccttgtggagaacggtgagactcactgatcccctgtgcgctggcagcaggaatgatgggaattagtgcagtgtgggatggagagagggctacactggagtgaagccagcagtgctgtgtgtgtgtgtgtgtgtgtgtacacatgcacgtatgtgcaggactcacacttgggattcagaaggtaaggggttggatggggattaggcagtgcagctgcctgctgtaagtggagcctgggggttggatgttgcttctggggagggccagggcctaagggtagtaacggactagaattcagaataaagacaggggctagggcaaaatgacggaagggttgggatggccccaaccagtgtcgtgccaggaccttaccctgtggttcacccccccagggctgctacgaaaaagtgaagctgtggtttgatgacaacaagcacgtgctaggcaccgtgggcatgtgcatcctcatcatgcaggtgaggggaccccgggcacctcgactctgggtggacgcaagccagagccaccgccaggctgcagggctccgagtgaggccacaggcaatgggcaaaggtggtgctcagtgctaggcggggccctgacatgctccgcatgagagctaaaggtgttcagtgtcccccgggtcaccagCTTTACAGGTGACATGTGGCACCTTGGGTGAAGCCACAGGTGGCCCCTGCCTGCACCTCTGTGTCTCCACctgctttcAGGGTTTTAGCTGAGGAGCGTAAATGGGATTTGATGTTGTTCAAGACAGTTATCCAAGACATCACTTCACGTGTTCTAGAACCACAGAGCTTGTTGGCATGAATGCCAATGACTCGAACACCCCCTTG

 

This mutation is a 2472 bp deletion beginning at Chromosome 6 position 127,965,063 bp and ending after 127,967,534 bp (GRCm38/mm10). In addition there is an 8 bp deletion (CTGCTTTC) 70 bp after the 2472 bp deletion that will not affect the results of the deletion. 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
39262 TCT GCC CAG AGG AGT AAC CT Common A
39263 TCA TGA GGT CTC TCT GAA GAT CA Wild type Reverse A
39264 TGG AGA CAC AGA GGT GCA G Mutant Reverse A
39265 Fluorophore-1 CTT CGT CTC ACT GAA CAC TGA AGT Quencher-1 WT Probe
39266 Fluorophore-2 TTC TGG TTT CCC CCT TTA CAG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
39262 0.40 uM
39263 0.40 uM
39264 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.