Protocol 31777: Probe Assay - Palm2<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

>chr4:57647918+57648007 90bp TTGATGGAGAAGTCCTGCTG TTCCCGAAGCACTTTGGACT

Mutant=  95 bp

Wild Type = 90 bp

Sequence

Wt Sequence:  GACAGATTTGATGGAGAAGTCCTGCTGGAAAAGATGTGGACcaaaaggtgtcatctgttgtcttctctccttcccacagtccaaagtgcttcgggaaaaatggctgctgcagggtgtaccagcgggaacagcggaggaggaggaagccaggcgcaggcagtctgaagaagatgaattcaaagtcaagcagcttgaagataacattcagaggtaggtagttcccagcctgcagtaaccactgctctcctgaaggggacgctttcgccgtgctgcgacttttccctggggctgtagattgctgtcctaaatagatactggtgtcggctaacttttCAGTGTGAGTTGCTGTTATTCCTCAGCGAATAGTACAGAGATGGTTTCCCCCACCAATGAGGTCTTTATTATAAGCAGTCTGCCCTCCCCCAAATGCTCTCCTGTAAGT

 

Deleted Sequence: caaaaggtgtcatctgttgtcttctctccttcccacagtccaaagtgcttcgggaaaaatggctgctgcagggtgtaccagcgggaacagcggaggaggaggaagccaggcgcaggcagtctgaagaagatgaattcaaagtcaagcagcttgaagataacattcagaggtaggtagttcccagcctgcagtaaccactgctctcctgaaggggacgctttcgccgtgctgcgacttttccctggggctgtagattgctgtcctaaatagatactggtgtcggctaactttt

292 bp deletion beginning at Chromosome 4 negative strand position 57,648,243 bp and ending after 57,647,952 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37627 TTG ATG GAG AAG TCC TGC TG Common A
37628 TTC CCG AAG CAC TTT GGA CT Wild type Reverse A
37629 CTC ATT GGT GGG GGA AAC Mutant Reverse A
37630 Fluorophore-1 AGT GTG AGT TGC TGT TAT TCC TCA G Quencher-1 MUT Probe
37631 Fluorophore-2 AGG TGT CAT CTG TTG TCT TCT CTC Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37627 0.40 uM
37628 0.40 uM
37629 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.