Protocol 31591: Probe Assay - Brms1l<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

Mutant=  139 bp

Wild Type = 141 bp

>chr12:55841784+55841924 141bp GCCAGGAGGCTTCAGTTATTG GTGCAGCGATGGAAACACT

Sequence

WT sequence (deletion in lower case, ^G^ is an insertion):

tggatgtccagtttcatattacaactttttttttttctgtctggactaacatcagaattttttgaaacagttttatcttttctgtttgtttttattttttgagatagggtttcatgtatcccaggctggcctcaaacatgctaagtggcgaatagtcatagctagtcttagactcctaatcctgcctgtgcctcccaagcgctggaatctcaagtgtgtgcctccaggcctgccctgaagttttatctttgatatttccttttttttttttaacagaaatggatgatgaagattgtgaaaggagaaggatggagtgtttagatgagatgtccaacctggagaagcagtttacagacctcaaagatcagtaagtactaaccagatggccctcgtcactgaatctcagtggcatcagttttaccagcacaggagcaattacagcgtttctgctgtgtttccttttgtgttttatgggaactggctgatctggccaggaggcttcagttattgtttacccgctagagacctcattagctcaacagagttttgaggccagtgaggtTTTGGGGGTGGATGGAGTAGGTGACTTGCATGGGCACGCTGCTGATTCGAGTGTTTCCATCGCTGCACACTGCTTCTGTGTGTGCTGTTCATTCCCTTGTTA

 

562 bp deletion beginning at Chromosome 12 positive strand position 55,841,295 bp and ending after 55,841,856 bp (GRCm38/mm10). This mutation deletes ENSMUSE00000654815 (exon 2) and 471 bp of flanking intronic sequence including the splice acceptor and donor. In addition there is a single bp (G) insertion at the deletion site

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37223 GAC ATT ACG TTA ATT CTA TGG TGG T Mutant Forward A
37224 GTG CAG CGA TGG AAA CAC T Common A
37225 GCC AGG AGG CTT CAG TTA TTG Wild type Forward A
37226 Fluorophore-1 CTG GAC CTG TTT GGG GGT Quencher-1 MUT Probe
37227 Fluorophore-2 CCG CTA GAG ACC TCA TTA GCT C Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37223 0.40 uM
37224 0.40 uM
37225 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.