Protocol 37455: Probe Assay - Aatf<em1(IMPC)J> Alt 1
Version 2.0

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

>chr11:84473300-84473398 99bp GGCCACTGAAGTCTTTCTAGC CTCCAGTGCCTGTCTCATGT

Mutant=  90 bp

Wild Type = 99 bp

Sequence

Wt Sequence (deletions in lower case):

AGGAGAGAAAGAACTGAGCTCATTCTTCAGAAGAGGGGAGGGCCTTTCAGGAGGAATTGCTTATCAGACTCAGCACCCTTGAGACCTGAGGGCAGCCTGTAGGATATACCCGAGGAACTTAATATCAGGGGCAGAGTTCATTATGGTAACAGGCAAGGAGTTTGCCAAACTTAATGACTTTAAGAACTGGCCACTGAAGTCTTTCTAGCTGCAGACACATTTCTAAGCACCTTCCACgtgctgctatcttgcttattccttagcgtcacatgagacaggcactggagtagtttccatggagttgatctcagaaagtactgactgtccccactcagaacttgtcaggagacagggagtgacagcaattggagagttgtaaatgactaattatcccttttctctgctttatttctattccaggtcacaaagcacttaaagccttgttaaggtcattggtggatcttcaggaagagttgcttttccagtacccagacacgagacatatagtaaatggggcgaaacccaatacagagaggtaaggaggaagcagagccatggcttgattattttcttctcatctgaatgaaaaaggaaggcgggtctttctcagacagatagatacgggatttgataaacgtaaaatagagtcgcttttatatttaacAGGTGGTCAGAACAGGACAGTTTAGAGTGCTTGTTCTAGTTTTTTTTTGTAACTGCTTATCTTTATAGCTTAAGGCAAAATATTTGCCTTCTCTTTTGTTCTATAATAGTGTACTGGCTAGTTTTGTGTCAACTTGACACAGGCTGGAGTTATCACAGAGAAAGGAGCTTCAGTTGAGGAAATGCCTCCATGGGATCCAACTGTAAGGCATTTTCTCAATTAGTGATCAAGGGGGAAAGGCCCCTTGTGG

 

A 427 bp deletion beginning at Chromosome 11 position 84,472,923 bp and ending after 84,473,349 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49582 GGC CAC TGA AGT CTT TCT AGC Common A
49583 CTC CAG TGC CTG TCT CAT GT Wild type Reverse A
49584 AAC TAG AAC AAG CAC TCT AAA CTG TCC Mutant Reverse A
49585 Fluorophore-1 CAC CTT CCA CAG GTG GTC AG Quencher-1 MUT Probe
51370 Fluorophore-2 TGC TGC TAT CTT GCT TAT TCC TTA G Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
49582 0.40 uM
49583 0.40 uM
49584 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.