Protocol 35140: Probe Assay - Cstf1<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

>chr2:172376297+172376396 100bp GGTCTCGGTGATCAGCCCTA CCATACCCTGCTCATCTGC

Mut= 113 bp

Wt= 100 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TGGCAGGTGTGAGGTCCTGGGTTGTCCTCAGCAGCACAGAATAAACCAAGTGTTGTTTTCCATTCTGAATGGGAAAGATGGAATAATGGCCACACTGCTGCTTGCGTTGCACTTTGTTGTCTGGTTAGAGcaagtgaatggctgtggaggcggaaatcccctttcccagcccactctgcccatggagtgcgcctcacatagcatggaatggacagggctcacgatccatccgctccaggatgggtctttgcatttacagatcttctcatgctcttctcttgattcctttagggatggagaacgatgacactgcggtccagtatgccattggtcgctctgacacggttgccccgggcacagggatcgacctggagtttgatgcagacgttcaaacaatgtcaccagaggcttccgagtatgaaacctgttatgtcacttcccacaaaggcccgtgccgcgtggccacctacagcagagacgggcagttgatagccacaggatctgctgatgcttccataaagatactggacacagaaagaatgttggccaaaagtgccatgccaattgaggtaccattcccgcctaccactgctccagacagcgtggctgtgttgtcaccagctctggtttgccccgtgggactgtgggcgtggatccatgcctctgtgccttgagcatctatctctccttttcttgttggtccaggtcatgatgaatgagactgcacaacagaacatggagaaccacccagtgatccgaactctttacgaccacgtggatgaagtcacgtgtcttgcttttcacccaacagaacagatcctggcctcgggctcaagggattatactctgaaattgttcgattattccaaaccgtctgcaaaaagagctttcaaatacattcaggttagacctaacaggagcacttgactttctgatacagtatttcaagtggtctcggtgatcagccctacctggtacttagataagatgccattgagGGGTCACTGGCTGTCCTTTGCCCAAGCAAGACTGCAGATGAGCAGGGTATGGTAGATCACACAGTAATCCCAACACTGAGGAGGTCTGCCACAAGTTCAAGGCTAACCTGCTGCATAGTCAACTCCTTGCCAGCTTGAACCA

This mutation is an 868 bp deletion beginning at Chromosome 2 position 172,375,477 bp and ending after 172,376,344 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
45364 GGT CTC GGT GAT CAG CCC TA Wild type Forward A
45365 CCA TAC CCT GCT CAT CTG C Common A
45366 TGG GAA AGA TGG AAT AAT GG Mutant Forward A
45367 Fluorophore-1 CCT GGT ACT TAG ATA AGA TGC CAT T Quencher-1 WT Probe
45368 Fluorophore-2 TTG TCT GGT TAG AGG GGT CAC T Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
45364 0.40 uM
45365 0.40 uM
45366 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.