Protocol 31720: Probe Assay - Tsga13<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

>chr6:30912310-30912419 110bp TGAGTTATGGGCACCTAAGTTAC CATCCAGGTGCATTACTGACTC

Mutant=  102 bp

Wild Type = 110 bp

Sequence

Wt Sequence: GGATATGATGCATCTATTTGAGTTATGGGCACCTAAGTTACTAACCTCTCGAGTTAAGTTCCCaaacttaaacggagtgagtctgtggacctgcagaaatgctcaggagtcagtaatgcacctggatgtcccactcccttttcaggtttcaagaagtcagtggatcaaagcctgtgggaaatgtctctattaggtttgagaaacaactaggtcaagatgatgaagaggtaagaagacaaaacaacagcaacagccttatatgtccctatgtcatctttccttgggattgcttaaactatcttacaagtagtgggcttgtaaacttcctatgtaaaagcaaaggtgaacaaacatttatatacagagtgaagttaatgtattccatttttgaaaaaggaacagagaagttgaggcggtaacAAGGGTATATCATCAACAAATCAAGACACAAAACTGAAAACCACTTA

 

Mutant Sequence: aaacttaaacggagtgagtctgtggacctgcagaaatgctcaggagtcagtaatgcacctggatgtcccactcccttttcaggtttcaagaagtcagtggatcaaagcctgtgggaaatgtctctattaggtttgagaaacaactaggtcaagatgatgaagaggtaagaagacaaaacaacagcaacagccttatatgtccctatgtcatctttccttgggattgcttaaactatcttacaagtagtgggcttgtaaacttcctatgtaaaagcaaaggtgaacaaacatttatatacagagtgaagttaatgtattccatttttgaaaaaggaacagagaagttgaggcggtaac

357 bp deletion beginning at Chromosome 6 negative strand position 30,912,374 bp and ending after 30,912,018 bp (GRCm38/mm10). This mutation deletes ENSMUSE00000289889 (exon 2) and 275 bp of flanking intronic sequence including the splice acceptor and donor. In addition, 64 bp before the exon deletion there is an indel consisting of a 4 bp insertion, ATAT, and a 7 bp deletion, CGCAAGA

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37488 TGA GTT ATG GGC ACC TAA GTT AC Common A
37489 CAT CCA GGT GCA TTA CTG ACT C Wild type Reverse A
37490 CTT CCT ATG GTA AGT GGT TTT CAG T Mutant Reverse A
37491 Fluorophore-1 CGG AGT GAG TCT GTG GAC CT Quencher-1 WT Probe
37492 Fluorophore-2 AGG GTA TAT CAT CAA CAA ATC AAG ACA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37488 0.40 uM
37489 0.40 uM
37490 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.