Protocol 38019: Probe Assay - Pop5<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

>chr5:115238266+115238412 147bp TGGGCTTTGCAGGTACGAG GCACCTTCTGTTCAGTTTTCG

Mut= 147 bp

Wt= 147 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

CTAGAAAAACGCATTAAAAACCTTAACCCTCTGTTCTTCATTTTAAAACATAATCCTCTATTCTTAACAGAAAACAGAGGGCTGGGTGTGGTGGGACGCGTTGGTAATGCCAGCGCTTCGTAGGAAAAGGCAGGGGATCAGAAGTTCGGGGTCGTCTTGGCTACACAGTCAGTCTGGGGTTAGCTTGAGGAAAAAAAGAAACCAGGAAGAGAGGGAGGGAGGATACTATATTGTTTTTGGAAACCACTTGAAAAGAACTCCCCTCCacaaaactgtcgaaatcccctcaaagaattcgcgtatttggctgtatgggaaaacctgtgctctgacttttaggtcccagatcgtgcgacactctcggactttaagtgcagggctgaccttaaacgttcacttgggggactctgagcgcacagaacttgtgtggaaaatccccagcgccggccggaggcgaacgcatgcgctcttacgcccgccgcgggaggcggagcgtggagattgacagggcacgctgcctgtccgagcctagtgccgccttcctcccgcatccggcgcggacgccatggtgcggttcaagcacaggtaagcagcgctgtccccgcgccgtccccgcgacccgggccgcgctcaccccgctgtcgccgcaggtaccttctgtgcgagctggtgtccgaggacgcgcgctgccgtttgagcctggacgaccgcgtgctgggcgggctggtccgcgacacgatcgcccgggtgcacggggccttcggcgccgccgcctgctccgtgggctttgcaggtacgagggcgctgcggatgcagtgcggggcgcgctttgccagggcgaggcccggccagtgtgtcgcgttttgtacgatagtggAGGGTTCTAGAAAATCGGAGTGCGGCGGGTCGAAAACTGAACAGAAGGTGCCGATCGCGCAGTGGGACAAGGCAAAAAGTTCAATACCCAGCCCCCGGTGTGTGTGCCGGGGTGGGTTGAGTTGGGGGATTCCGTTTACTGCCCGTTTTAGTGGAAGATGGGTGTTGAATTCACTAGGCTAAATGAAAGTAGCCATGTAGAATTCCTAAGAGCCATGATTTGACAGGTCACGTTAGCACGTTTTAAAATGTCAAATGTATCCCTCAAAGTTTAAACCCGGCTGCGAGCTCGAACA

This mutation is a 611 bp deletion beginning at Chromosome 5 position 115,237,751 bp and ending after 115,238,361 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
52786 TGG GCT TTG CAG GTA CGA G Wild type Forward A
52787 GCA CCT TCT GTT CAG TTT TCG Common A
52788 AGT CTG GGG TTA GCT TGA GGA Mutant Forward A
52789 Fluorophore-1 CAG TGT GTC GCG TTT TGT ACG Quencher-1 WT Probe
52790 Fluorophore-2 AAG AAC TCC CCT CCA GGG TTC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
52786 0.40 uM
52787 0.40 uM
52788 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.