Protocol 33757: Probe Assay - Dhx58<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

>chr11:100702981-100703100 120bp GGCCATTGATCACATCCTACA AGCGGAAGAGACGAGCATAG

Mut= 121 bp

Wt= 120 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 CTGATGGCTCGGAGCCACGACCTGCTCATCTGTACGGCAGAGTTGTTACAGTTGGCACTCAACAGCTCTGAGGAGGATGAACACGTAGAGCTCAGAGGTGAGGGGGAAAGCCGTGACCTGctcacaggacgtgctcctgggcaagcaagcctccttcctctagcccagcctcgctttcattcctacctgctccctgtctgggacccagcccaaagtagggaagggaagaggaaaacttcagagaaaaatgagggcttcacaaaagtgctatgagaaagaagcagcagcagcagccgtccactgtctctcatgtgcccccagaattctcgctgattgtggtggacgagtgtcaccacacccacaaggacaccgtctacaacaccatcttgagccggtacctagaacagaagctgaagaaggcagagcccctcccccaggtcctgggtctcacagcctccccaggcactggaggggccaccaagctccaaggggccattgatcacatcctacaggtcagctgagcccctcccaccatagatctttgcttgtcttgcctcccagtCCTACCAAATATACTCTTCATACTATGGCTATGCTCGTCTCTTCCGCTACCTTGAGCATATGAGGCTTGTGGGACCCTTTGAACTTACTATTCCTTTTGCCTGGAAATGCC

This mutation is a 442 bp deletion beginning at Chromosome 11 position 100,703,029 bp and ending after 100,703,470 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
42134 GGC CAT TGA TCA CAT CCT ACA Wild type Forward A
42135 AGC GGA AGA GAC GAG CAT AG Common A
42136 ACA GTT GGC ACT CAA CAG CTC Mutant Forward A
42137 Fluorophore-1 AGC CCC TCC CAC CAT AGA Quencher-1 WT Probe
42138 Fluorophore-2 CGT GAC CTG CCT ACC AAA TAT AC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
42134 0.40 uM
42135 0.40 uM
42136 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.