Protocol 33158: Probe Assay - Psmd2<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

>chr16:20654328+20654457 130bp GGCAGAAGCATCTTTCTTTGA AAAGTTCCATGCTAAGCCTCA

Mut= 133 bp

Wt= 130 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 

TTTTTTTTTTTTTTTTTTTTTTGGTTTTTCGAGACAGAGTTTCTCTGTATAACTCTGGCTGTCCTAGAACTCATCTGTAGACCAGGCTGGCCTCGGACTCAGAAATCCACCTGCCTCTGCCTCCCGAGTGCTGGGATTAAAGGCGTGCGCCACCACTGCCCGGCAGAAGCATCTTTCTTTGATTCCCCcattgtttattactcttgagttaggttagcttcagagggagctagcaacaccaactttagggtttttttttttttttttttttgaggcttagcatggaacttttctgtatagagactagtatatattgaattttataggtgaattttgtggacagtaatcttgattctgtaatggcacatgctgttttcctagtgttttgcagctgacatcatctctgttttggccatgaccatgagtggtgagcgggagtgcctcaagtatcggctcgtgggctcccaggaagaactggcgtcatggggccatgaatacgtcaggtaaggcattttctttatggacatgagaagggacttggggtcattattctgtttgtttgggaaactgaatgtattgaatcccccagacactgaattccttgactcccagcaacatgcttccctaatgagcaagtctatctatccatagacatctggcaggagaggtggccaaggagtggcaggagctggatgatgcagagaaagcacagcgggaaccactgcttaccttggtaaaggaaatcgtcccctacaacatggctcacaatgcagagcacgaggcttgtgacctgctcatggaaattgagcaggtggacatgctagagaaggacattgatgagaacgcctacgccaaggtctgcctctatctcaccaggtgagtgagtacaggagagcaggtagcactcgtcttccagggagtcttagggttcctcttgaaggaagagactttgtgactccttagtaatcctagaacatgctacattgccttctgtatagtaggcattcagtgtgtatacttgggaagggatttaaataagttacatgagttgcacgtcctgaaattgtactacttttgactttgtagttgtgtgaattatgtaccagaacctgagaactctgccctcctgcgctgcgccttgggcgtgttccgaaagtttagccgtttccctgaagctctgagattggcactgatgctcaatgacatggaactggtagaagacattttcacttcctgcaaagatgtgtatgtagagaaattggcaaaaggaaagatgagtctgtgtatgtgctttgtgtaaaaatcagagacgtagcagtttctgatgcgacacagaggcttttctgtctcatagtggctaagagtgagcaggtgtgaccgcttcccccacagggtggtccagaagcagatggcattcatgctgggtcggcatggagtgttcctggagctgagtgaagatgtagaggaatatgaagacctcacagaaatcatgtccaatgtccagctcaacagcaacttcttggctttagctcgggaggtgagggtctcctcttggtcctgaagggttttggccgaagttctgtcattctacctccctgactaatacctttcaatagactccctggttaaagttgctattcagtgggagaacagaagggacagctaggtaggggagtactggggaagcagttgtgaccctctggctttttttcagctggacatcatggagcccaaggtgcctgatgacatctacaaaacccacctagagaataacagtgagtagctctgtgggcattggagggactgttaattgttctaattgtattggGAATAACAGCTTAGACAACCCATTTCTAAAGCACTTCAAATTCTTATGGAGACATAGATTAATTTAAAGTTGGAGACTGGGCTAGCAAGATGGCTCAGTGGTTAAGAGCGCCGACTGCTCTTCCAAAGATCCCGAGTTCAAATTCCAGCAACCACATGGTGGCTCACAACCATCCGTAACGAAATCTGATGCCCTCTT

 

This mutation is a 1640 bp deletion beginning at Chromosome 16 position 20,654,355 bp and ending after 20,655,994 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
40779 GGC AGA AGC ATC TTT CTT TGA Common A
40780 AAA GTT CCA TGC TAA GCC TCA Wild type Reverse A
40781 CTT AAC CAC TGA GCC ATC TTG C Mutant Reverse A
40782 Fluorophore-1 CAG AGG GAG CTA GCA ACA CC Quencher-1 WT Probe
40783 Fluorophore-2 CCC CGA ATA ACA GCT TAG ACA A Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
40779 0.40 uM
40780 0.40 uM
40781 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.