Protocol 35712: Probe Assay - Trim68<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

>chr7:102681802-102681958 157bp CTGTGGGAGTTAAGATGACTCT CACTGGAGCACTCTAGGCTGA

Mut= 168 bp

Wt= 157 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TGTGTTGGGTAAGACTCAAGCTTCTGCCATGCTGATATTCCTGGCTTTGGACCACACTTTCAACAGCAAGTTCTGGGAAATAATAGAGAACAGGACTAATGAGAGGAGAAATAGTCCTGGCTAGGAAAGTCAGGTTATATATCTTTATAGACGCTTGAGGACTGTGGGAGTTAAGATGACTCTCAACCCAGAAAGCctccctgcatgtcacatgggcagaggctgtgccttccgtggcagtccttttcctcttatgttatcccagccagctgcctgaggaacattgggaagcccctgtcagcctagagtgctccagtggcctcctgatggggcacgctcatgatccttctgtctcttgcagtggaaactccagcaggctctggagcatctgaggaaagaacaggaagaagcctggaaactggaagtcagtgagaaggaacaggctgccatctggaaggcaggttgcacttgagaagggggctcactcacatcacccctctcttctagagccgacggtggagtcttcctcacgtctgtgaggcagagtgagggccttcagcctcccttttcctaaggccacataacttctcaggtccagcttatagctttggggtagcagtagcctctgatttcatatcttcacgtatgaggaaggaatggcgccttgcacacgtgagctcctaggagtatagcttatagagtggtgatctaacagtttctgtctcactttcctgagtgacgacggcacagctgctaagactttggttgcctaaatattaggaacccccctcagaaatatatttgaaaccaataggcataaaactctctgaacttttggtccatgtgatctaaaacaatacaataaacataaacacacacaaacacacacaaaacaaagaagaaaaactatccagcagtcttgttagcaaccatcagtgtaaccatctgactggttttctgaaaatgagagctagtccccatcatgccaagataatatcatgtaccaggatggtggtacatgcctagaagtcagccttgggaggcaagggcagagaactgctacagtttcaagtccaacctggtctacatagtgaataacagtccagggtggcaggggctagacactggagccctgtctcaagaagaaagaaagaagataacctagattctgttgcagttgataaaagtagtgcagacagattagaggtcactcatccagacatctgaactgaaagtgctcccaaatccaaagtgtcttatagccaacatgctaccaaaagtagaaggaagtttcgctgaatgataggttatactcagaatgcaagcacactaaaatcattttacaaaattactttcaggttctctgtacaagatacatgtgaagcatggatgaatttcatgttcagagttggatctcattgccaaagtatctctttatattatgtaatctaaaatccagaaaagttccaagtctgaaatactctggtcccactcattatttgtaagaaaatacactacttatgaaaggaccaaaaataatgcctggggccatggggagttaggaacagaaaagttagaccctttgtctttgccttcataacacccagaacatggttgtagcaaagcatatccatcgtgtgtgtgtttgcacatgtcagtgtgctccatcccacgtgacccttaccctaaaaagaccagtgctaattccagtcttgactccagacacagatggaaagacgaaagcagagcatccgatgggagtttgaaaaatatcggcagttattaaaggaaaaggagctgccctgccagcaggcagaagaggaggcagctgcagctcaggctagcctcgagcaggagaagggagagacagcaagcaaactggagttgaggcgcgaggcgatcatccggcagagccaggtcctgtggagcatgattgtggagctggaagagagatcccagagacctgtgcgctggatgctgcaggtgagcaacagccttccgtgggatgcctgagcgagaactggagacggaggctccaggggaaagtattagctagttcttcacggaccctatcatcattctgtccaccagctgaggaccctgtctgccttcagctgacacatgctgctctcatcctgattgggtaaaagacctgacccttaattttctcataggccagaagtaggagtacaccgggtacttactctatagcttggcagcattctagtcccttccatcctggtcatcttgagatggagtctgaaggtgttccatggagccaggtctgctctgctgtcctcccatgagcttctccccacctctaaagtctgtgtaaccctgtgagaaagcccctcccagagtttacgcctgtctagtgtctctgacgcaccgcacattcatgggctgcattttttttctcctagggtattcaggaagccttgaacaggtacgttcccatccctagagactctgtgaaaccctacaggagatagagaggcctggccagagccagggatggagaaggaatgtagactgagtacctacagaaggcagatgctcaccgtCTTACATTAGTGAAAGAAGATTTGTCTCCTTTTTTGCACTTAATTGAATTCATTTCAGATTTTTCCCAATAAGAATTTTAAGTAAAGCTGAAGTATGTTACTTCATGTAAATGGTCCAAAAATTTGATGCGTGAAACTTCTAGCTCAGAAGATCACCCTTTCTCCTTTGTGTCACATCTCTTTCTCTCC

This mutation is a 2368 bp deletion beginning at Chromosome 7 position 102,679,556 bp and ending after 102,681,923 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46838 CTG TGG GAG TTA AGA TGA CTC T Common A
46839 CAC TGG AGC ACT CTA GGC TGA Wild type Reverse A
46840 CAC GCA TCA AAT TTT TGG AC Mutant Reverse A
46841 Fluorophore-1 CTG CCT GAG GAA CAT TGG GA Quencher-1 WT Probe
46842 Fluorophore-2 AAA GAA GAT TTG TCT CCT TTT TTG CAC T Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46838 0.40 uM
46839 0.40 uM
46840 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.