Protocol 33837: Probe Assay - Pfdn1<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

>chr18:36450992-36451077 86bp GAGTAAATCAGCGCACCCTTC CCCAATTTGAAAATAAACCATTC

Mut= 88 bp

Wt= 86 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TCTGACTCCCAGTTGTTAGGATTATAGGTGGGAGACACCAGGTCTGGCTCTACAGCCAGGATTTTGAAATAACGTGGGTGTTTTGGATTCTAGTTATGCAATGCAGATGTAGTACGTTGTTTCCTGCTTTGGGTCTCTGGGTTTATCTCCTGAGTATtttatttatcccttttattactctctggctacttgataaatatggctttttttttttttaatgcacatttgtttttgtgttcatattggtcatgcggtctatggatagggtcaatgaatagggtacatttagaatttgcatagtgtgatcattctgttgacttttatctatgcttatcttggtaaatcttacagctttgtcaagttagcatgatctgttttaacaaatctaaatttgacttgaaagtttaatttcctaaatcaagcacttgtttttctcaggccttcacagagcttcaagccaaagttatcgatactcaacagaaggtgaagcttgcagacatacagattgaacagctgaacagaacgaaaaagcacgcacaccttacggacacggagatcatgacgctggtagatgagactaacatgtacgaaggtgtaggaagaatgtgagtaaatcagcgcacccttcacggggctctcttaggagaggcTTCAGGGGAATTAAATGGCAGAATGGTTTATTTTCAAATTGGGTTACTTAAAAAAGAAAACTTGGGTTAAAATAGAGAAATAACTTGCAAAAGCTATCTTTAAAAAAAAAAAAGGCATAAAACCCCAGGTGTGTGTTTTAAAATTTTATGTTTTTCACTCTCTCT

This mutation is a 493 bp deletion beginning at Chromosome 18 position 36,451,035 bp and ending after 36,451,527 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
42302 GAG TAA ATC AGC GCA CCC TTC Wild type Forward A
42303 CCC AAT TTG AAA ATA AAC CAT TC Common A
42304 TAC GTT GTT TCC TGC TTT GG Mutant Forward A
42305 Fluorophore-1 ACG GGG CTC TCT TAG GAG AG Quencher-1 WT Probe
42306 Fluorophore-2 CTG AGT ATT TCA GGG GAA TTA AAT G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
42302 0.40 uM
42303 0.40 uM
42304 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.