Protocol 33153: Probe Assay - Ap2a1<em1(IMPC)J> Alt1
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

>chr7:44909874-44909992 119bp TGTTTTGTGGATGAGGAGACAG CCTTCCAGCCTATCTGTTGC

 

Mut= 137 bp

Wt= 119 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case):

 TCTATTTCTGACGTATTGGTTGCAGGCGAGGTCTGGGTGCACAGATGTGACCCACAGTGTCTGCTAGTCTCTTTATTACCTATAGCAATGCAGGgtacacgaggGTGCTCACGATGCAGGGCCGAGCCAGCCTGCACATTTTGCCTGGCCCTGCCAGTTGTTTTGTGGATGAGGAGACAGAGTCTCTGGGGCAGCCAGGGCAGTTTCCAAGTGctttggggttgctctggctgcctatgtaatatcctggtcacgtggcaacagataggctggaaggtgcactggggccatgtagccctgactgccccacggtgtcccacagggacagcatggacagtgtgaagcagagtgccgccctatgcctactgcgactctacaaggcctcgcccgacttggtgcccatgggcgagtggacggcacgtgtagtgcacttgctcaatgatcagcacatggtgaggtgcccggccgccccaccctcaagtgcctgaagcccatgtcttgccttctgccctgacattccaacccagacctcctgtccccagtgtgtatgccctgcccagtttccaagaaaccttcttccttcctcgataactccttggctgcctgggactgtcctcatctctgatgcagtaccttaacaagatttgcctatttctgagcctagagagcccatagcaaggccagtggactgactgccttgcaaagactgaccccaccctagactcccattagcccaccctgctcctgactcctgagctgacttaccctgatgccagggctttcccagtgacacccagcatgaagtccctcacgcctgagctccttgcagggagtggtcacagctgctgtcagcctcatcacctgtctctgcaagaagaatccggatgacttcaagacctgtatctccctggctgtgtctcgcttaagccgggtgagtgtgctgtcacactggccgtgggagggtctggctcccctgtgtctgtgatatctgtctctcctcccttccctaaaaggcaagccagcctgtgcttagaagccaggactctgcattggcttcctcgggctgtgtgcccccaggcaaatggcttcacttctctttgcttcttttcctgtgtggttcaggggctggtgtcagtgcggtctgcagCATGGTTGCTGTGGTAAGGTGCTGACAGCTCCCCTGCCTGCTCTTCTTCCTCCTGTTTGTCTACACATCCCTCCTTTGCCTCCCACCACCTCCACCCTTGTCCCCAGAGCGCCGCCCCGAGCCTTCTGTGTGCCTGGCTCTCTATGTCT

This mutation is a 934 bp deletion beginning at Chromosome 7 position 44909004 bp and ending after 44909937 bp (GRCm38/mm10). In addition, there is  a 10 bp deletion ( GTACACGAGG) 109 bp before the 934 bp del that will not alter the results of the exon deletion.  

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37545 CCT TCC AGC CTA TCT GTT GC Wild type Reverse A
39982 TGT TTT GTG GAT GAG GAG ACA G Common A
39984 Fluorophore-1 CTG GCT GCC TAT GTA ATA TCC TG Quencher-1
39985 Fluorophore-2 AGT GCA TGG TTG CTG TGG T Quencher-2
40758 GAG GCA AAG GAG GGA TGT GT Mutant Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37545 0.40 uM
39982 0.40 uM
40758 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.