Protocol 34609: Probe Assay - Mrgbp<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

>chr2:180583500+180583597 98bp CTATGTACGACATGCAGGCACT TGTACCACAGCTGACAGGATCT

Mut= 95 bp

Wt= 98 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 CAAGCCTGTCGGTGAGCGCCGCGCCGCGCACCGGCGTGGGGGTGGGGCGGGATAAACCGTCGGGCGGGCTGGCGGAGCCTGCGACCTCCCACGGGACGTGGCCTCCCACTCTGGGGTGCCCTACTAGTCTAAGAGCCTATGACTTTCCTGCCCGAGAAGCCCTCTCCCTGgggacactcctgacttccctgcccttgagacactcctaccccagggaacgcctccattcccccccacccccccacctctgcagccccctgtccttggacccgcctactatacacaccctagcccctggaacacccacctgccttgtggcacctctcttcccctttgacatgtgactcctgcaacacttgcacctgcgggtttgcccactcctgcccctccttcttcacccatgtcctacgcacagaggtgctgtcctaaccctcaacccactgtcctacctctcaggggtgaatcggcacttccacatgatttgtatccgagacaagttcagccagaatattgggcgccaggttccatccaaggtgatctgggaccatctgagcactatgtacgacatgcaggcactggtgagttggatcctggcctgcccatttacgggctgaccgtgGAGTCAAGTCCCAGATCCTGTCAGCTGTGGTACAGGACAGAACCCTCTTCTCACTTGGGGTTTGGTATGCAGAGCAGAGGTGATCTCAAAGGATGAGGCTCCTTCCTCAGAAAAGCTTAAGTCCTGTTGTGAAAAGCACAGACTAGCGATGATCTCTGACTACT

This mutation is a 449 bp deletion beginning at Chromosome 2 position 180,583,115 bp and ending after 180,583,563 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44005 CTA TGT ACG ACA TGC AGG CAC T Wild type Forward A
44006 TGT ACC ACA GCT GAC AGG ATC T Common A
44007 TCT GGG GTG CCC TAC TAG TCT Mutant Forward A
44008 Fluorophore-1 CCT GGC CTG CCC ATT TA Quencher-1 WT Probe
44009 Fluorophore-2 AAG CCC TCT CCC TGG AGT C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44005 0.40 uM
44006 0.40 uM
44007 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.