Protocol 34340: Probe Assay - Aamp<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

>chr1:74280664-74280768 105bp CTGGCAGCTCTAGAAAGTGG ACAGGACACAGTCAGGCACT

Mut= 102 bp

Wt= 105 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

GGCTGGCTGAGATGAGCATGGTGCTGGGGTAGGAGTAGACAGAGGCACAGAGAAGATAGGCTCTGGTTGCTTAACAGTGGGGCCAGTAGTATCTGTTCTTGGGAAACGGGAGTGCTAGTCCCTGGagctgcagcaagtttggcccccttctttgctgaggtcctcctactttcttttctgctcagttgcctttaaacagtgactagatgcatgggtcacttgtccccaacatttaggtattttttaatgttcttttttgccacctttttaaaggttatatagttgggttggccttggatgttggcaaggctggcctcaaacttggtacatctcttaggcaaacttgagtttaaaacctgcctacttctctttgctgcaggccacaaagactctgtgacatgcgctggtttcagtcacgattccaccctagtggccacaggggacatgagtggtcttttgaaagtatggcaggtagacaccaaggaagaagtctggtcctttgaagcaggagacctggaggtgagatccttggagtgagcttccactctttgggaccttaagtgagagggatagcggcagacggatccagatctagcctggagaagtttgcctagagccatagcagcccagctctagcaatgtatctcttcgcctgtaaagttctcgctgaccttagaaggtcacaccttagatgaggctcatggacttaggtaggctgggtagtaggccctgcctgctgatcctccctctctgatcagtggatggagtggcaccctcgggctcctgtcctgttggcgggcacagccgatggcaacacttggatgtggaaggtcccgaatggtgactgtaagacctttcagggccccaactgccctgcaacctgtggccgcgtcctccctgatggtgagagcagttatctcttcctgggggtacaggtggatccagtgacttggggttacttcagtcattgcagtctcctgccctatacttggtcttacctgtgactccagggggaatttggggaccccctacactatcctttctgctcattctttccttggcgtgtctgagtgtcctctgtcctctggttatccgtgccttggtccttagggaagagagctgtggtagggtatgaagatggtaccatcaggatctgggacctcaagcaaggaaaccccatccatgtactaaaaggtatcagagggggcggggtgttttccttggctttgagggaggagcctgagccaggtacaggactcaaaacctctgtccacctgcttgttctagggactgagggtcatcagggccctctgacctgtgtagccaccaaccaggatggcagtctgatcctgactggctctgtagactgccaggccaagctggtcagtgccaccactggcaaggtaagtagcctgggcttctttgtctggaccttctgcacctcccctgtcttttccctctttgtccaggtctgtgagcctcttttcttacctgtaggtggtgggtgtgttcagacctgagacagtcgcctcccagcccagcctgggagagggggaggagagtgagtctaactccgtggagtccttgggcttctgcagtgtgtgagtgtgagcagaaccttggcctggagacccaagagactgagggaggaggtggctttgaggaggcatctaggaattgagaccaaggaggagggaaggtatatgaatctaggacctcaagagccttctttgtgtcacttgcttcccagcatgcctctggccgctgttggctatctggacggaaccttggccatctatgacctgtctacgcagacactccggcaccaatgtcagcaccaggtgtgatgtctggggcttaattcctacttaggggctgttgtgctggctaaggggcagcctgtcctctgtggctcctcagggtgggttggggtcctctcttctcccttctgacttctgccctactatgttactgtgtcacactccgggacagtcgggcattgttcagctgctgtgggaggctggcactgctgtggtgtacacctgtagcctggatggtgttgtacgtctctgggatgcccggactggccgcctgcttactgactaccggggccacactgctgagatcctggactttgccctcagcaagtaagcattatgggctggcagctctagaaagtgggtctggtgtgaggtagcacaggtcctttgtgGAATGGGAGGCGATTCCTAAGGGAAACGGGACCCAGTGCCTGACTGTGTCCTGTAATAAAGGCATGCTGGGCCGTCCATCTGTGCAGTCACAGTCCTAGAACCCAGCCCTATCCCAGCGATTGGCAGTGAACTACCCAT

This mutation is a 2087 bp deletion beginning at Chromosome 1 position 74,280,718 bp and ending after 74,282,804 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
43353 CTG GCA GCT CTA GAA AGT GG Wild type Forward A
43354 ACA GGA CAC AGT CAG GCA CT Common A
43355 TGG GGC CAG TAG TAT CTG TTC Mutant Forward A
43356 Fluorophore-1 CTG GTG TGA GGT AGC ACA GGT Quencher-1 WT Probe
43357 Fluorophore-2 CTA GTC CCT GGG AAT GGG A Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
43353 0.40 uM
43354 0.40 uM
43355 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.