Protocol 37471: Probe Assay - Plscr5<em1(IMPC)J> Alternate 1
Version 2.0

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

>chr9:92198517+92198632 116bp CCTCGAGCTACAAACCCAGA AGAGTGGCTGTTGGCAGGA

Mut = 120 bp

Wt = 116 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case):

 AGAAGCTATAGCTACAGGTTAGCAAGGGCCTGTTACCATTTAGTTTGCTTTTGTGAGCTATGATTCTTCATCTTCAAAAAAAAAATGCTACTTGTACAATGTATCTGCGGGTCAATTATTTAAGCCAAGTTATTCAgttatgtctccgggcaaccacaacattcttccatgttccttcttccctgactgagggcactgaatagtgaaaacggggaaaatgcgccaaaatgagagacatcaatcaaggtcaaaactgacacctaaatcaaattaaaaagaaaaagaaaagaaaaaagaatgaaagaaaaagaaaaagaaagaaaggaaggattatgtaaaatcaaaagcaacctaaactttaaatatccaacaagatatttatcttctcttgcagattcccagaaccaaaaccctagaagtctgcctgggtttcttcctcgagctacaaacccagatcaaaactctcaggaggcttcttccaatcccaggaaacaggcaaggcagcagccgggtgtctctcagccaagcagtttcctgccaacagccactctccctcctggtctagagtatttaagccaggtttgcatgacttcactttctactagagtgttttctttaaaagagcggtttcttgtttttcaaccaataatttaatggaatacaaaaatgcatgaaaatcatgaagttataaaagactgattttattgacttctattatttagaaagacttaatccactttattgccacctatccttatttacttgacagagtctcaagactaaacagtcTTGACTTTGTCATGCAACACGTTTATCTGCCTCTGCTCATGGCTCAAGGTGTTAAGATAGATATTCTCAAGGAATTATCAGACGAAACCACATACAGCTATTAATTAAGCATATTTATTGAACACTTGCTATATTCAAGGCTCGTAAGAAGCATAGCGCCTATCCCTTGTCACTCAAACATTTTGTAAGTTAATGATCAAACAAACAAACAAA

This mutation is a 953 bp deletion beginning at Chromosome 9 position 92,198,268 bp and ending after 92,198,820 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49628 CCT CGA GCT ACA AAC CCA GA Wild type Forward A
49629 AGA GTG GCT GTT GGC AGG A Wild type Reverse A
49630 TTG TGA GCT ATG ATT CTT CAT CTT C Mutant Forward A
49631 AGA GGC AGA TAA ACG TGT TGC Mutant Reverse A
49632 Fluorophore-1 CCA GGA AAC AGG CAA GGC A Quencher-1 WT Probe
51386 Fluorophore-2 CTA CTT GTA CAA TGT ATC TGC GGG TCA A Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
49628 0.40 uM
49629 0.40 uM
49630 0.40 uM
49631 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.