Protocol 35095: Probe Assay - Bend6<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

>chr1:33878456-33878568 113bp TGCGTTATAGCAGCTGTGTGT CAGACAACAAAAGACACTTTGC

Mut= 114 bp

Wt= 113 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TAGAAAGTCAGATTCCTGTCTTCTATTTGAAAAGGGAACATGAACCTTTTCGTGTGTCCTGTCCTGAGTTCTGGAGGGTGGAGAACAAAGCTGGGACTATGCGTTATAGCAGCTGTGTGTGGAGGCAGACATGCCGAGGTctgataagaggcattggcatacagaacatgaggtagctgttgaaggaactgcaaagtgtcttttgttgtctgttgtgttttatattttagagagacccatattcaggaaatgctttcctgcctggggagagttccagcgatgaggaaactcccttaatggaactgtccaaggaggaactttgtaacaaaatcgaaagcctgaaagaaaaactaagaagcatccggaaggaaaacagccggcttcgacagtctttggtcatgctgcaaggtaaaacctcaaaaggattggcattttagataaaagggacaaaatgtctaaaatatggtacaactgcagatcaacagttaaaaatggaaaagaaaatcagttcccaagagagctcaaaattacacttcagaaattagtggatagctaaatttaaatatggacatatccggttattgcagagataatttagattgtcttgcaatgtcccaaatattgtattcacttagtagtattcaacagattgctgaagtgaacttaagctacttacatgctgggcctcaGAGGGGGAAAATGAGTAAGGTCAAATAAACACGCTGTGGTTTCCAGATACACTCATCCCACCCTTCCTACCAGGACAGTTCTAGAAAGTGGGAACCGTGTGATCTGCCGAGCCACTGGGGTTACTGGCTCCACAGGCTGCACA

This mutation is a 549 bp deletion beginning at Chromosome 1 position 33,877,979 bp and ending after 33,878,527 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
45242 TGC GTT ATA GCA GCT GTG TGT Common A
45243 CAG ACA ACA AAA GAC ACT TTG C Wild type Reverse A
45244 CTG GTA GGA AGG GTG GGA TG Mutant Reverse A
45245 Fluorophore-1 AAG AGG CAT TGG CAT ACA GAA C Quencher-1 WT Probe
45246 Fluorophore-2 ACA CGC TGT GGT TTC CAG AT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
45242 0.40 uM
45243 0.40 uM
45244 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.