Protocol 38191: Probe Assay - Swt1<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

>chr1:151410810-151410928 119bp TGCAAAGTCACCAGATGTCA TTGGTGCTCACAATGGTCCT

Mut = 119 bp

Wt = 119 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

GCACTGAGCCTCTAGCCCTGTTTCTTATATTTCTCAGTAGTTTAGAATGAGCCTAgttaactctaatgtaatgatatactgcaacagaatgtataacaaatataactaacctaagtttctgtgctgctttatagattgaatgtagaagttttcattcctaaaaagagactaaaattcagttcttcgacccaagcggtatcatatttaaataataatcaaatgacttcacacagttgttcatcaaatggaactaaagatactaaagttaaagattgtaaattgacaaatattggaagtaagcttaattatgaaattaaaaaccatagccgcattaaaattaccaaagatatgaaatctaaacctgtagaccagacaaaggaaaagaattggccttctttgcttattcagaagaagatgaaagaactcaagaaagaaaaaaataataaagacagttctgaagaattggagaaatgcaagaaaaatcaccttcctcagaattataattttagcaatatgatcaaggaatcatttgaatcaggaagaaagaagattagcttcaaaattccaaaaaaatcctctactaccctccagaagcttgtcgaagaaaagatctttactatagattctagcaagtcaaagagtaagcaggaggagaagcaacacttgcaaagtcaccagatgtcattaaatctggccaggcacaaaactgaaaactcattttcagattctacacataagcagtctgtttgtgagaggataagaaaggaccattgtgagcaccaaggaacgagtagttccgattctaaggaaaaccatgcccaggtaatgtataaaatgaagggtttggtttttagtaaaagtatttaaaatactcaggttttagttgcttctgttatgatgataatcacagaggactgattctaacttagtcatgtagataattgtgtttgtgtcctatattattttttgtcattctagcttccttaggataatactgataccacctgGTTTTGGAATCATAGTGCAATATCTGGGTTCTTGTGCTCTGGGACATAGAACTGGACAGAGACCTG

This mutation is a 952 bp deletion beginning at Chromosome 1 position 151,410,586 bp and ending after 151,411,537 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
53252 TGC AAA GTC ACC AGA TGT CA Wild type Forward A
53253 TTG GTG CTC ACA ATG GTC CT Wild type Reverse A
53254 TGA GCC TCT AGC CCT GTT TC Mutant Forward A
53255 TGC AGG TCT CTG TCC AGT TCT Mutant Reverse A
53256 Fluorophore-1 CTG GCC AGG CAC AAA ACT G Quencher-1 WT Probe
53257 Fluorophore-2 CTG GGT TCT TGT GCT CTG GG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
53252 0.40 uM
53253 0.40 uM
53254 0.40 uM
53255 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.