Protocol 32423: Probe Assay - Mprip<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

>chr11:59727279+59727362 84bp TAGGATCTTGGCTGCCTGCT AACAGTGTCCTGGCTTGCAC

Mut= 86 bp

Wt= 84 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 

AGCCTTTGGGTCTGTCTCCCAATCTTAGAGCTAGTGCTCAAAGATGTGGCTCTATTTACCTACCTCCTTACGTGTTTTTTTGTTTGTTTGTTTGTTTTTTTTTTTTCTATTTTGAATCTACTATTCCCGAAGCTTTTCCTCACTGATCTGAGACTAGGATCTTGGCTGCCTGCTCTCCCTagcattcctcactgagcactgtcctgacccctcctgcggtgcaagccaggacactgttctccatgtagaaaataaagtccagagggtgtgtcctctacataagtccagcttggcttgccccaccgtggatgggtgggtaggaagtggtgctgtgaaggctgaggactgaaggcaaggggtgggtcctgctggtggctgtgggacctgacacaccaccccttttgagtgacttgttttgtccttgcagcccaccaccctccctcagggcaccatcaacatgaaccagtgcacagatgtggtggatggggaggcccgcacagggcagaagttctccctctgcatcctgacacctgacaaggaacattttatcagggcagaaaccaaggagatcatcagtgggtaagttggccagagccacaggaggcactgtctgtttgctcacccatttctggggactccagggcttctaggtcttccttggaactcttcactgattggtcgtcctcccaccccacacctcatcctcaccctgtgcctccttggcactcagcacacagtgctgctcctgccttcccctcaagtaaaataagccctcttttctaggctgtgtatcaggcagcctgcgtggcggcccctttgagaaacgtcccgtgtaGCACCAAAGCCTCCAGGTGATTCACATGCATGTATTTTCGGTCCTCAGTGATTAACCCCATTCATTCTCTACGGCTGGAAGAGGGCAGGTGTGTCATCAAGGCTCTGTCTGAGGTTGAGGGGTATAGGCTGTGTATAGCACAGGGTAGAGGGAAGTAAGGACATGAA

This mutation is a 655 bp deletion beginning at Chromosome 11 position 59,727,305 bp and ending after 59,727,959 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
39075 TAG GAT CTT GGC TGC CTG CT Common A
39076 AAC AGT GTC CTG GCT TGC AC Wild type Reverse A
39077 TGG GGT TAA TCA CTG AGG AC Mutant Reverse A
39078 Fluorophore-1 ATT CCT CAC TGA GCA CTG TCC T Quencher-1 WT Probe
39079 Fluorophore-2 CCT GCA CCA AAG CCT CC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
39075 0.40 uM
39076 0.40 uM
39077 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.