Protocol 31716: Probe Assay - Mfsd4b4<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

>chr10:39894595-39894685 91bp TAGCGTGCTGGCATCCACT TCAGGTGGCCAACACTCAC

 

Mutant=  103 bp

Wild Type = 91 bp

Sequence

Wt Sequence: AAAGGTTATGACTAGAAGGCCACTGGGGAGTCTGTGGCTTTACTGCTGATAATGTGCATGTTTTCTTAGTTCCACTGTGGTAGACTGTCATCTCCCTCCtgtggttttgttacagtgggaaagctactttttggtcagtgttccagcgatagtaagaggctctcttgcttttaactatagggactaagtgttgctatcctggggcccacctttccagacttggcgaagaacgtgaacagaaacatcagcagcctgtctctgattttcgtgggccgggcctcaggatatttatgtggctctatgattggaggagtcctgtttggctgtataaatcattttttacttttgggtaagtaaacaccgctcttctttgtttctgtcaacaaatttacaactcagctctaaatggggaagatggtggatacctgagccaggtagcgtgctggcatccactgtgggagctgggaagtctggactgattccctggGAACTCGATGGGCCCTGGGTGTGAGTGTTGGCCACCTGAGCAATGCTGACTTTACTGTGATATAGGCTCCACTCCAGGCACAGGCACTGGCTGACCCTGAAGCTCTGCTTATCTGGGGTAGGGGCGGGGCTTCTGGGGCCACGGGGGAGGAGGA

 

Deleted Sequence: tgtggttttgttacagtgggaaagctactttttggtcagtgttccagcgatagtaagaggctctcttgcttttaactatagggactaagtgttgctatcctggggcccacctttccagacttggcgaagaacgtgaacagaaacatcagcagcctgtctctgattttcgtgggccgggcctcaggatatttatgtggctctatgattggaggagtcctgtttggctgtataaatcattttttacttttgggtaagtaaacaccgctcttctttgtttctgtcaacaaatttacaactcagctctaaatggggaagatggtggatacctgagccaggtagcgtgctggcatccactgtgggagctgggaagtctggactgattccctgg

388 bp deletion beginning at Chromosome 10 negative strand position 39,895,021 bp and ending after 39,894,634 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37468 TAG CGT GCT GGC ATC CAC T Wild type Forward A
37469 TCA GGT GGC CAA CAC TCA C Common A
37470 GGC TTT ACT GCT GAT AAT GTG C Mutant Forward A
37471 Fluorophore-1 AGC TGG GAA GTC TGG ACT GAT Quencher-1 WT Probe
37472 Fluorophore-2 TGG TAG ACT GTC ATC TCC CTC C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37468 0.40 uM
37469 0.40 uM
37470 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.