Protocol 33843: Probe Assay - Wdr6<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

>chr9:108576501-108576611 111bp TGCTGATGTGGCTCTTTACC CCAAGCAGGTTCTGGACTCT

Mut= 111 bp

Wt= 111 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 AGGTCCGGCTGAAGTGGAGGCACGGGTTTGGTAGGATGCTGTGTCCTGGTATATTCTGAGTCTGTATGCTAAGGAGTGGCCTGCTCCACTCATGCGTCCTAGGATCAGGTGCGTGCGTGCGTGTGTGGTGCTGATGTGGCTCTTTACCTCTGTCTAGGCGAGGGGCctgatttactggtgtacaacttggaccttggtgggcatctccgaatggtgaagagagtccagaacctgcttggtcactttctcatccatgggttccgagtgcgaccagagcctaaaggagacctggactctgaggccatgatagctgtgtttgggagcaagggcctcaaagttgtgaaagtcagctggggtcaaagccatcttcgggagctctggcgctctggcctgtggaacatgtccgactggatctgggatgtccgctggatcgagggtaacgtagccgtggccttgggccacaactcggtggtactgtatgacccagtgatagggtgcatgctgcaggacgtcccctgcacagacaggtgtaccctgtcctcagcctgcctggttggtgacacctggaaggaactgaccatcgtggctggcgcggtttccaatgagctcctgatctggtacccagccactgctttaacagacaacaaacccgtggcccctgaccggcgggttagtggccatgtgggtgtcatctttagcatgtcatacctggaaagcaagggcctgctggcaactgcttcagaagaccgaagtgttcgtctctggaaggtgggggacctccgggtgcctgggggtcgggttcagaatattggccactgctttgggcacagtgcccgagtgtggcaggtgaagctcttagagaactatctcatcagtgcaggagaagactgtgtctgcttggtgtggagccacgaaggcgagatccttcaagcctttcggggccaccggggccgaggtatccgggccatagccactcacgagaggcaggcctgggtggtcactgggggagacgactcaggcattcgactctggcacctggcaggccgagggtacccaggcttgggcgtctcatccctgtccttcaaatctcctagccggccaggtgccctcaaggctgtgactctggctggttcctggcgagtcctggcagtgactgatgtggggtccctgtacctctacgaccttgaggtcaagtcctgggagcagctgctggaggacaatcgctttcggtcttactgcctgctagaggcagctcctgggcccgagggctttggactctgtgccttggccaacggggagggtcttgttaaggtggttcccatcaacacccccaccgctgccgtcgagcagaaactgttccaggggaaggtgcacagcctgagctgggcccttcgtggttacgaggagctgcttttgttagcatcgggccctggtggggtgatagcttgtttggagatctcagctgcacccactggcaaggctatctttgtcaaggaacgttgccggtacctccttcccccaagcaagcaacgatggcacacatgtagtgctttcctgcccccgggtgacttcctcgtctgtggggaccgccgtggctctgtgatgctattccctgtcagaccatgtctattcaaaaagcctggggccggaagcaaggctattactgcagctgaggcacctggagctggtagtgggagcggtgggtctgagagtgtcccaacaggaataggccccgtctctacactccattctctgcatgggaaacagggtgtgacctcagttacctgccatggtggctacctatacagcacagggcgggatagctcctacttccagctctttgtacatggcggccacctccagccggtcctaaggcagaaagcctgtcgaggcatgaactgggtagctgggcttcggatggtgcctgatggaagtatggtcatcttgggtttccatgccaacgagtttgtagtgtggagcccccggtcccatgagaagctgcatatcgtcaactgtgggggagggcaccgctcctgggccttttctgatactgaggcagccatggcctttacctacctgaaggatggtgaggtcatgctctatcgggctctaggtggctgcatccggcccaatgtgattctccgggagggtctgcatggccgtgaaatcacatgtgtaaagcgtgtgggcacagtgaccctgggccctgaatttgaggtacccaacttggagcatcctgactccctggagcctggcagtgaggggcctggtctgattgacatagtgataacaggcagtgaggacactactgtctgtgtcctagcacttcccaccaccacaggctcagcccacgccctcacttctgtctgtaaccatatctcctctgtgcgagccctggcagtgtgggctgttggcaccccaggtggcccacaggatactcgaccagggctcactgctcaggtagtgtctgcagggggccgagcggagattcactgcttcagcgtcatggtcactccggacgctagcacccccagccgccttgcctgtcatgtcatgcacctttcatcccaccggctggatgagtactgggaccggcagcggaacaagcacaagatgatcaaggtggaccctgagacCAGGTAAGGCGGCCTCCCTGTCATGGGCGGGGCGTGAGAGGGTTATAGGAAAGTCCTGACTCCTGACTGCAGGCCCCACCTGACCGGTGTGTACCCCCTACAGGTACATGTCTCTTGCTATTTGTGAGCTTGACAACGATAGGCCTGGCCTCGGCCCTGGCCCCCTTGTGG

This mutation is a 2,470 bp deletion beginning at Chromosome 9 position 108,574,104 bp and ending after 108,576,573 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
42316 TGC TGA TGT GGC TCT TTA CC Common A
42317 CCA AGC AGG TTC TGG ACT CT Wild type Reverse A
42318 CCT GCA GTC AGG AGT CAG GA Mutant Reverse A
42319 Fluorophore-1 CTT GGA CCT TGG TGG GC Quencher-1 WT Probe
42320 Fluorophore-2 AGG GGC CAG GTA AGG C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
42316 0.40 uM
42317 0.40 uM
42318 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.