Protocol 36365: Probe Assay - Eif5b<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

>chr1:38018742+38018883 142bp GACAGTCATGCTTCCATAAAAGG CTTCATTGTTTTCTGTAGGCTAAAAAG

Mut = 136 bp

Wt = 142 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case):

 GTGTGGTTGGATTTGTTCCTATTTTTTTTCCTCTTTTGTTTTCTTTTAGATTTTTGCTTTGTGTATGAGTGTTTTGCCTGCATATAGCTACATGTACCATGTGTGTGCCTGGTGCTCACAGAGGTCAGACGAGAGGGCATTAGATCCTCTACAGCTGGAGTTACAGATGGTTGTGAGCTGCATGTGAGTGCAGGAGCTAAACCTGAGTTCTTTGCAAGAGCAACAGTTGCTCCTCACTGCTAAGCTACCTCCAGACTCATACTTTAATATTCTTGTGAAACAAGACAGTCATGCTTCCATAAAAGGTATTGAGgtgagcactgagtgaagtgctccacactgaggagccccatgacctttaagggatgacagtctgttgatgtttctaataagggatactttttagcctacagaaaacaatgaagaagaatctgcctcaaaacaagataaaaagaagaagggacaaaaaggcaagaaaacaagttttgatgagaatgatagtgaagaattggaagataaagattcaaagtcaaaaaagactgccagacctaactcagaggcacccctttctgggagtgaggatgctgatgattctaataaactttctaaaaaaggtaaaaaagcccagaaatccacgaaaaaacgggacgggtcagaagaggatgaagacaatagcaaaagaagtaaagaacgttcgagagtaaactcctctggtgaaagtggtggtgagtccgatgaatttttgcagtccagaaaaggacagaaaaaaaatcagaagaacaagtccgttcctaccgtagacagtgggaatgaggacgatgactcttccttcaaaattaagacggtggcccagaagaaggcagaaaagaaagagcgcgagaagaaaaagagagatgaagagaaagcgaagttgcggaagatgaaggagaaagaagagctagagaagggcaagaaggagcagagtaaacagagggagcctcagaagagacctgaggaggaggtgttgacactcagagggactcctgacacaggagctgcctcggaagagaaaggagacactgctgcagctctggaaggttggtcatggccacagaaggctctggttcatgtttgGTCTAAGTTGTGGTCATACTATCTTGTGACTAGTCCTCATACACATGTGTGTGTGTGTGGGGGGGGGGTCCATCTCTTTGTGTGCAGAGGAGGCTGGAGGTCGATTCCGCTGTCACTTTCCATGATCTGTTTTTAATATTACACCATTTCTCTACGGTCTTTCTGTATACCTCTCTTTTAAATTTATGGCCTTGTTTTTTTCCCATGAACTGCTGTGAGGTTGAGTCCCCCGGAGTGTCAGAAACTGTATGTATAAAGCCTCACCCAAATGGCTTTCTGAACAGCAGTTGAATAGAGACACCAACAGATAGATACAGTAAAGCCAGTGCCTCAGCTCTCTGTAGAGAACCACAGGCAACCAAGGAATGCTGAGATAGAGCATAAGAAATCGTCTTCCATCTGATTTTTTTGAGACAGGGTTTCACTGAACATAGAGTTAGTATACTGGCTGGTCAGGAAACAGTGCTGTCACGTCTGGCTTTTCTTTTTATTTTTTAAGATTTATTTATTTTATGTA

This mutation is a 800 bp deletion beginning at Chromosome 1 position 38,018,772 bp and ending after 38,019,571 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48532 GAC AGT CAT GCT TCC ATA AAA GG Common A
48533 CTT CAT TGT TTT CTG TAG GCT AAA AAG Wild type Reverse A
48534 AAT CGA CCT CCA GCC TCC TC Mutant Reverse A
48535 Fluorophore-1 TTA AGG GAT GAC AGT CTG TTG ATG Quencher-1 WT Probe
48536 Fluorophore-2 AGG TCT AAG TTG TGG TCA TAC TAT CTT GT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48532 0.40 uM
48533 0.40 uM
48534 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.