Protocol 31538: Probe Assay - Cracd<em1(IMPC)J> Probe Alternate 1
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.

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

Mutant=  137 bp

Wild Type = 136 bp

>chr5:76859302+76859437 136bp GAATGTGGTCCCAGTTAAACG GCACAGCAGAGCTTGGAACT

Sequence

 

 

Deleted Region: tggatgttaagagcgccgtgcctggcatgttggttagggaggtgtgggaaaaatgctaatcgcgcttcccttcccaatgccaggaccgacagaatgaaccgggcagctttgagagtcagtcctccctggaccagaacgggcagctaggagaagacaagcacatttggcacggagaggaaccagaacctttggaatcccacgaagagaaaagactgcacgaagagtattggcgggaacttgaagccaagtgcaaacgacaaaaagccgaagccgctgagaagaggcgccaggaagagcagaggcggcaggcgctggagaggaggctttgggaggagagcctcagacaggagctgctggaagaggaggaggagggggaggaggaagaagaggtgaaggaggagggggaggagggcgaagaggtgggcctgcagccacgggcagggaaggtacctccagaggaagggcaccagtctggtccggaggaacagcgctgtacagagcaagagctggggggagccgacgatcccgcccgcctggaggcagaagagcgcaggcgtgagcaggaagaggccgagaggcaggcggagaaactgaggcagcgcgaggctgagaggcaagaggaggaacagaagcagcgcgaggccgagaggcaagaggaggaagcgagacaatgcgaggccgagaggcaggcggagaaactgaggcagcgcgaggccgagaggcaggaggaggaagcaagacaacgcgaggccgagaggcaggcggagaaactgaggcagcgcgaggccgagaggcaggaggaggaactgaggcaacgggaggaggctgagaggcaggagaaactgaggcagcgcgatgctgaaagccaagaggaggaactaaggcagcgcgaggcggagaggcagcgcgctcaggaggaggacgccaaggggatgctgcaagaggaggaggaggaggccaagcgcatagaagagctgaaaggaaaggagacaccagagccgctggtggaggaaggtccgcagagtccagaaggagagagccagccttggctaacggacgatgctgaccagcgaagcccactgcagcgggaccttgagaagccaggggaacgggaacgagaagacctggaatccgcagggcagagagagatcgcggaggagcctagaggagagggcgagccggccgagcagagcggagacttggacgcgcactgcggtggtgtggacggggaagggaaagaaaccgctcagacagacagtccccagccgcaggagagacagatggaaggaacaccggccccggaggagaatgaagctactgcggcagacatagaccggaaggtggaggagctccggtggcaggaggtggacgagcggcagaccatgccccggccctacacattccaggtttcctctgggagtaggcagattctcttccccaaagtcaacctgagcccggtaacacccgctaaggacgcgagcttggccccagcggcccaggaaccgcccgcgccccgcggtgccgcctcacacgccctgccatcagcgctgagcatcccacacacggccattttggtcaccggagcgcagctctgcggcccggcggtcaacttgagccagatcaaggacacagcatgtaagtccctcctggggctgtcagaagaaaagcgccctatggacgtccccactgtggagtccagggccggcagtgggaagtccaggcctgccccagagtcaccaagcaatgcggccgcgctagccgagtgggcttccattcgatccagaatcctgaagaactcggagggcgaccaacgtggcgacagagagccggccagagccggggacgagccagtgcctcgagcgcggtgcgattcccgagggaacgtgcgcaggacgccgccagtcaacgccaagttctccatcatgcctgcctggcaaaagttttcggacagcggcgcagagaccttcagacaaagcctggacggggagagcggaaggaaaaagccggggctagcacccagcgaagaaacagcaccccagcctcatgcagcggctcagcaagaggtaagccaggagccaccagacaccacagacggatgcaaatttgccaaagaccttccatctttcctcgttccaggcctgccttctccgcagaaagctgcgtcccgcacggagtccacgaccactttggacagcgagaccacgagtgacgtgggaaacccagaccctgcgatgccaggaggcgaggaaaaggcctcgccctttggaataaaattgagaaggaccaactattccttgcgcttccactgtgaccaacaggccgaacagaagaagaaaaagaggcacagcagcacaggagacagtgtgggtggggccacccccgcaactgggagtgtctctggagagagcgagcctgaagccacgttcctcaagcatggcccatcccttccccaggagagaaagccagccctatcccctcggaaggactctgctgaaagccattctagcggtcactatgttgctgtggcccagtctggtctaccaccggccagtggccagaccccagctccagagcaagacagggcagtaagcaaaatgccatccatgcagaagccggccctggctcccaagcctgccagccagaccccaccttcatccccactctccaaactgagcagaccccacttagtggagcttcttgctaggcgagctgggaagctcgactccgagcccagtgagactgccaaggagagcagtgataaccagccaccctcaccatcactgcccgaggagctgaaggggcagaagagggatgagaaagatgtgccagaaaagaagcctgcttccccacctctgccagcgggccagcaagagagaccttcgctaattcctgaaacaggtagaaaaggtgtgtggctgggggttggatgtggcttcattcgatggggagggaattgagagggagcccacgggaaactggttctgggaacacctcagttaggaacaggtttgcccccaaatcagtccctttacattttatacattatttgaattggaatgtggtcccagttaaacgcagatattgttagccagtctgtagagatgatcaaaagtagaatttccttgaccagtgtctgcccttcagtctgggtag

3153 bp deletion beginning at Chromosome 5 positive strand position 76,856,248 bp and ending after 76,859,400 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37100 TCC ATG GTG ATT CTA AGT GCA G Mutant Forward A
37101 GCA CAG CAG AGC TTG GAA CT Common A
37102 GAA TGT GGT CCC AGT TAA ACG Wild type Forward A
37103 Fluorophore-1 TCT ATA CTC AGG ACC ACC TGC C Quencher-1 MUT Probe
37104 Fluorophore-2 ACC AGT GTC TGC CCT TCA GT Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37100 0.40 uM
37101 0.40 uM
37102 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.