Protocol 37123: Probe Assay - Hsfy2<em1(IMPC)J>Alternate 2
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

 


>chr1:56637303-56637408 106bp TGTGAAGTCTGTGTGGGTAAAG CCGGAAGAGGTCTCTAAATCTG

 

Mut = 119 bp

Wt = 106 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case):

CCAATATAATCTGCAGAGTACAAAGCTGTTCAGATGTGAAGTCTGTGTGGGTAAAGTGTACAGGATATGgctgaagcaccttcggaaatgcaggctgtttcccctaacagtctatcaacagatttagagacctcttccggcgagccttggtgtgacaacacaggtcagaaggactcagacttatgggctattattgaggaaagtgcatttcaggttttggctcagcgattcttgataaagaggccaccgcataccctctgtgcctcagagccagatgaagacagtaacttattttcaatgaccttccccagaaaactctggaaaatagtgggaagtgacaaatttaagtctatttggtgggatgaggacgggacttacatagtgatcaatgaagagctcttcaagaaggaagtgttggaaagaaaggcccccttcagaatatttgaaactgatagcatgaaaagtttggttcgacaacttaatctgtatggattcaggaaaatgagacagaacttccaaagatctgcttctctccccgactttctggctgaagaaaaaggaatttctgcctcatgtaagttacaattctaccagaatccaaactttctgagagactgtccccatcttatagaaagaatgaaaaggagaattgggattaaaacagtttctcgtggagctgctccttcccttcctgatttcaagaacaaacacttcagaccagacttggtcaagatggatgaccatagtttgggtgtagctgtccaaaccagtgagagaaacttgttgtccagctctagtatttcaaatgtatatctgagacagaagccttctcttgctcaagggggttctgatacgatggatgtcatcagaagcgacttttctcttgcaacgccaagttccttcagaccacctgaagaaattctaacacatcggcctgctccattaaatgaggtgtcctctctttgtatggactcacagagaatcttcactcaagcaaacgacagcactgtgaatttcattataacctctattactcaaaaccgcggggacatgtctcacttgtggaatagttgtattgaaatgcaaggagagccttcctttcagcctgggtttcctcacttttcatccagttcatctacttattctgactcaaaagcaaagggcgaacctgagttaccgatacacaaggaaagagtagcatctaccacgttggcatcaacatcaaaccatcactcatcttcttgagGCCATCAGTTTTATGATTACCAACAATCTACTTGGTTCTTTAGGAAGACATTCATCATAGCAGAGATTAGAATTCATGTTGACAAACACCTGGAGTGTACCTGAGATTATTGTAATAGAACAATAAAACTCTATGTGC

This mutation is a 1177 bp deletion beginning at Chromosome 1 position 56,636,197 bp and ending after 56,637,373 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49335 TGT GAA GTC TGT GTG GGT AAA G Common A
49336 CCG GAA GAG GTC TCT AAA TCT G Wild type Reverse A
50577 TGT CAA CAT GAA TTC TAA TCT CTG C Mutant Reverse A
50578 Fluorophore-1 ACC TTC GGA AAT GCA GGC T Quencher-1 WT Probe
50579 Fluorophore-2 ATA TGG CCA TCA GTT TTA TGA TTA CCA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
49335 0.40 uM
49336 0.40 uM
50577 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.