Protocol 35096: Probe Assay - Bpifc<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

>chr10:85995611-85995752 142bp GGGGAAACGTCTCTTAGAAGC AACATCCCTGGGCTTGTGAC

Mut= 148 bp

Wt= 142 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 CAAGACGTGCCTTGCGATTATTGTAGCATCCTTTAAAGGCAATTTTTCATCGTTTCCATCACAACATTGGACATGTAATGAAAACTTCTCACAGTAGCCCTGTGGTCTCAGGCAAGCTTCTGTGCctctcttaagactggagtttccgtatgtgaagccaagactacacctcttttgtggggagggggcatgctgcttcctcctctaacatctggtctttctcaatgctgtgtctgtagggttaaagatactgtgtcaccaccatcttccttatgatcaacaaacacttcttgcatccctcacaggtcttcaagttgggatgaaggtactggagcaattggcaaaggaaatcgtcatcccagatttgaacggttctgagtctcttaaatttctgaagattgactatgtaaagtacaacttctcaaagtaagagagtggagcatgttgacttatatcgttaggggctcaaaaccagtggagaaatctatggcagccctaacttagttatctttttttttttttctgtttcatctaagtcctgacttctattcacaagtttgcctcatagctcaatatatctaatactgtgtcgaccacctgatgcatatgtagctgaaaggggaaacgtctcttagaagctcagttcatttaagctaatttaacctcagccacattaattttctcatcTTTGAGGTCTTGTATGGGGCTCTAAGCTAAGCACATTTCATCTATACAACTTAGTCACAAGCCCAGGGATGTTTGGGGAAGGGGAAATGGACATTTTAATGGTAACAAGAATCCAATAAGATTACAAA

This mutation is a 562 bp deletion beginning at Chromosome 10 position 85,995,684 bp and ending after 85,996,245 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
45252 GGG GAA ACG TCT CTT AGA AGC Wild type Forward A
45253 AAC ATC CCT GGG CTT GTG AC Common A
45254 CGT TTC CAT CAC AAC ATT GG Mutant Forward A
45255 Fluorophore-1 AAG CTA ATT TAA CCT CAG CCA CAT T Quencher-1 WT Probe
45256 Fluorophore-2 AAG CTT CTG TGC TTT GAG GTC TT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
45252 0.40 uM
45253 0.40 uM
45254 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.