Protocol 33399: Probe Assay - Ppfia1<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

>chr7:144510166-144510252 87bp AGCTCTGTGGGTATGCAAGG GTCACATCACTGGGCACCTC

Mut= 104 bp

Wt= 87 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case and insertion with carrots ^g^ (TGTCTGCTTTGTTACAGAACCCAGTGCTACTAGATTGCCCCCTGTGGCTGGATCT insertion):

 ATCTCTGCAACCCTAGGTCCTTGGGTGATCTGTTTGAGACTAATAGCATGGGTTTCTTGTAAGAGACAGGGCCAGCTGGGATCTCTGTGTGGGTTGGGGTGGAAGATAATAAGGACTGGCAGCCTGTGGGCTAGGACAGGTGGGCCATTTATACAGTTTTTACACCCTGGCCTTGCCC^ccttttaacttgtctatcaccaggagtactgcttttcatttactagggcatccatcgatttggagacccaagttctgcttactctaggactcaggaagtgttctcagaattgaaacgcatcttataggggccgagtccatgtgagagagttgccatggaagctgtttattgttttctaggaccaacttgtcgtgactattgaagccctgaaggctgagctggagcagatgagactgagaggtccttcactccatcatgggtatggcggtcacagatggactaggtttcagctctgtgggtatgcaagggcttgctgctgtctctgcgggc^GTGGGAAGAGGCCTCTGTGCTGACTGAGGTGCCCAGTGATGTGACAGAAGAGAGAACATAATGGGAGCAGAAGGACAGGCCTCATAGAAGAGTCCCACTGCAGGAGGGAGTGCCATGGGCAGCTGCTCAGAGTCCATTTCATAGCTTCGGT

This mutation is a 330 bp deletion beginning at Chromosome 7 position 144,510,211 bp and ending after 144,510,540 bp (GRCm38/mm10). In addition, a 55 bp sequence corresponding to Chr 7:144512416-144512470, from the upstream intron between exons 11 and 12, and inserted into the deletion site. 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
41326 AGC TCT GTG GGT ATG CAA GG Wild type Forward A
41327 GTC ACA TCA CTG GGC ACC TC Common A
41328 GCC CTG TCT GCT TTG TTA CAG Mutant Forward A
41329 Fluorophore-1 CTT GCT GCT GTC TCT GCG Quencher-1 WT Probe
41330 Fluorophore-2 TGG CTG GAT CTG TGG GA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
41326 0.40 uM
41327 0.40 uM
41328 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.