Protocol 36316: Probe Assay - Mbip<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

>chr12:56339872-56340000 129bp CAAGTGAAGGCCGAACTCT AAAACTGCTCCCTTGTGAGG

Mut= 118 bp

Wt= 129 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case; and insertion with carrots ^g^ (G insertion):

 TTAGGCATGTGTTTGTGTGATATGTAACAAAGAGAATACACTTTCTGGACTGTCGTAGTTGATCTTCGTCTGTTGAATGCCTAGTTGGGATTCAGTAGGATTCTGACTAGGGCAGATCTATTTTTGGATACCTTCCAGTGGTCTTTAAAATATCAAAGTATCAAAAGAGGTTTATCTTTCATGGATGGCTTTTCAAAGATGGCCCCAGGAACAGAATTAAAATAATAAGACATGTTTTTACTTTTTATTTTCAAAACTGTGCCTTTTGGCTTTTTAAAATGGCAACTGGGTAAAGCATTTTATTATGTCATTGAAACTTAGTAACTTTGAGCAA^aataggcatgcattactccatagaaatcttatttttgtgataatcccagatatacagcttaatcgtgtaatttttgtgatgttgctggtttgcctctttgaagcttaacctcagagatgatgtggtgaaaattacaatcgattggaacaggctccagagcctctcagcatcccagcctgcactgctccttactgcacttgaacaacacgttttgtatttacaggtaaatttcatattagaaatatttactggatattgaaaatagaaattacctatagttaaattaactggagtatttttcagttctacctcaacattctgttaacagtattattaataaaaggttattaaaatccatattgagtctaggtctgttaatttcactagtgatcggaatagttatgagaatatagcccaataggccaatgtgggtatgagattgaataaatgcaattctattcaattctaggctttgctgaaaattcaatacagttcatctatggtgggttataattaagcaataagacacggcaggtgtgagtcatgctatgataatgattccatgccttgggtgagtttggaggctcatggagtgctttcagtggttcaagaagtggcattatcccagcatgatacatcctggagtgtctattgcaattaaacagtttcagcatagtttttaaaaagaaagtaatttctgtgacattaatgtttcatcttagtgagtagccagtcactatttcatcttaataatttgaaataatttatcctttattggaagtcaatattgaattatttataatggtattgttacatttttaatatttcagtttaagaaatgaacagtattataaccttgctgttgttgtttaacatctctgcgagtgtaattgatgggagttggaggttttctctcatcaggataagcagaactgctttttctggtcactttaccctgttgaagatgttagaagatttcagaagagtactttctgggatgtttagctgtgtttcatgataaaaataacagttaacactttgtttaaagggcaagtgatgagctatagaactcaaatgtggaccatatccaggtggggatgacaggaaaggaagtgaatttccattatgaagacattttttttaacctactgtagttggaatgatctgtttccaacgtgtatagcatgttttgttttctttctttctatctacctatcttccttccttccttccttccttccttccttccttcctttctctttcttctccccgtgtgcattttcttttgtgaaatttcatttcatgtcccaataagatatcatactatgacagagaaaggctgactattcactcagggtaacacctatatacttcatatccatgctctctcattcattttggccaactgtactttgaagtggagagtctcagcgatacagtgataatgcagggctttgctgagtgagcactgccctcctgctaaggagggagaacactcagcctagtgttctgaatgacatgttaattaagaggcatccaggccagagtctgaggctttgatttagcacaaaataaaagatgaatgacagaatgatttcctcaaagaaaaatttaaatgtcctcaggttttctttcttttcttcttttttaaaaataaatgtacagtaaatgataagggcaatgtacagtagtagaaaacagcaaacctgtgtgtgttaagatctgtgtgtccttgtatgccagctttttatgtttaataactgataatattcatttctgtagcagcagcttagataaatctatcactagctgagatgagtttgaacctgaacagatgtaactgtgttctttccagccttttttagcaaaacttcagtctttaatgaaagaaaactcaacagctacagagataagacagacagaagcagagaccaagagtgaactaagagccatacatcccaccgaggacctacaagatgaagggaagcccaaagactgtgatgtaggagatgtgaaaaagacacaaaacctttttgacccagaagtagttcagatcaaggctggaaaagcagaagtgagtgtggacaggtacacgggtcagggtaacattactttgaaaagttctgttgtgtttagattgttaaattatttgttgttttagattgacagacgaatatctgcatttattgagagaaaacaagctgaaatcaatgaaaacaatgtcagggaattttgcaatgttattgattgtaatcaaggtaactacaagtttttttttttaacagaattttaaacatcttgacaactgctataatattttgttttaaaaacatataacattaagaacaagattctattttgcaggtgtttagttgataattaaggtagtagaattttcctttatttagtaatgggctctgttgccaaactgattctcattttggttacataaaaaatccaagtgaaggccgaactctagagacagatgtttaccacaggttcaagaagatcagagttcctttagaaa^GTGAAGGTGAACCAGCGAACCTAGTAGCACTACTTGCACCCCTCACAAGGGAGCAGTTTTCTCCTGACTCCTGAGTTTCATGGTGGATACACCCAGCTTGCCATGAAGTCCTGTGGAGCCTCAGCCCCAGGCCTCTCCCTTTTCACACTGCTGTCTCACCTTTGCCGTTTCGCCTCTGTCTCCCTTTCCTCTTCGTTTGGGGCTACAGTGTTTCCAGGTCTGCAGAGGACCTGCAGAACCCCATAGACTGTGTAGATTGCCACAGATCATAACCAGTTAGCTTTGTAATAACCAGACTGTAAG

This mutation is a 2527 bp deletion beginning at Chromosome 12 position 56,339,932 bp and ending after 56,342,458 bp (GRCm38/mm10). In addition, there is a 1 bp insertion (G) at the deletion site.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48409 CAA GTG AAG GCC GAA CTC T Wild type Forward A
48410 AAA ACT GCT CCC TTG TGA GG Common A
48411 AAT GGC AAC TGG GTA AAG CA Mutant Forward A
48412 Fluorophore-1 CCA CAG GTT CAA GAA GAT CAG AGT Quencher-1 WT Probe
48413 Fluorophore-2 AAC TTT GAG CAA GGT GAA GGT G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48409 0.40 uM
48410 0.40 uM
48411 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.