Protocol 34813: Probe Assay - Wdr18<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

>chr10:79966827+79966919 93bp TACCCAGGTCACTGGCATC TGAGCCCGTTAGGCTGACA

Mut= 92 bp

Wt= 93 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 TGCAGCTTCTCACAGCCCAGTGCCCCGCCCCTTGATGGCATGGGGTGCACACAGTCAAACTTTCTGTATCCTGCCTCTACAAGTGGCCCTGACCCTGTGGCAAACTGGACATGGGACACCATTTTAACCCACATGGCCTTAGCCTCCcaagtagtggaaggacaccgcccccattgcccctctgtgtgctgagctggaggctcacacattttgagccagcaggggcctcttctggggacagggtagaatcccattattgtcagacactgaggacacagccagagctggccaagtgcacagtgatgagggcctgcagccttcctggttccatatgccaatggccctggagcaggtggacggggttcttatgggttggcatattctgtgttgcccctgaccttcagtgcccggcctctcacaggtctgcaccgggaacctcctggtcatcctcagccggcactaccaggacgtgtcctgcctgaagtttacaggggatggcagtcattttgtctcagcgggcaaggactgcttggcgctggcctggagcctctgcaggtagggcatgtgccagagatctacccagaaaggctatggaggtctcagggcaggcaataggctggtgactactgaggtatcccattcttgggggcttggggaggcaggccaggggctttccacgtggcctcactgctgttgtgtgcccagtgtgctacaggcagacccatccaggatccttgccccaagacatgtctggtcccagcacacgctcccaatcacagacctgcactgcggcttcggaggccccatggcccgggtggccaccgcttcattggaccagactgtgaaggtagcatccccagcctcgacccatgaattcctggcagccactggatctcagctaggagggagggagactggcatctcccttgccttggctgtttgggctccagacagcccataatccctatgcccgaccaaggacacccagagatggactgtccagcctgtctgctggaaattcccactcgtccacgcctcagtggccagatgcccttgagtgtgtagggttgtttgtttgttattggcacagtgttagagatggaacccaggccctgcatctgctagaccagtgctctacctgagcctcacctcagccctcttttatcttctagttttaaacagttttttgcttgttttttgagactgtctcattgtgtggccacagcttgcctggaacttggtttgtacaccagattggctagggattcacagacccccttgcctctctctgcctaggagcacagggattgagggcaggattggagacatattccctggctggggtgcctgtgattctgtgggggttcagaggctggtagcatgggcatgatggtgcgatggcccccatgcctcctggccccccgacttggctgtgtgcctggatgtccacagctccaggcttcccattcctgcagctctgggccatctcctcgggggacctgctgctgtctgtcttgtttgacatgggcatcacatctgtgaccatggacctggccgagcaccacatattctgtggaggcagcgatggctccatcttccaggtggacctgtgcagctgggtgagtggccctctgggactgagggagcccacagtcctttgccctacatcccaggcacttaacgacctgtctctacccccagcccggactgagggagcacagcttccagcctgagcagaacacagggaaggtcttcaaaggacacaggtagtcacctcaggaggtggggttagcacagtgcccacaccgtgaagcacctaacccccatcctcctctcccaggaaccaagtgacatgcctgtcagtgtcaacagacggcagtgttctgctctctggctcccacgatgaatcagtgaggctgtgggacgtgaagagcaagcagtgcttgcgcacagtcaccctcaaaggtgtgcccgaggcagcgtggccacacagggacaaggcagggccctgtcctgccagccaggcccctgatgttgtgttattgtgtccttcaggcccagtgaccaatgcagccatcattctggccccacccagcatgctgaaccctgaattcaggcccagcctgcccctgccccacttcaacaagcacctgctgggggctgagcatggagatgaagcccagggtgggggcctgcgcctgcagctagggctccacctgcaggtaggacccacagccactcttctagaacattctatactccagagttccttctgctgctgctggtgtgcctgtctggcatcaacagcttctgaacccttgctacccaggtcactggcatccactgctgctcttgccatccgtccccacaacatacgcctgtCTGAAGGCATACTGTGTCAGCCTAACGGGCTCAGCTGACTTGTCACATTGCTCTCAGTGAGTGGCCAGAGAGAGAGTCAGAGCAGCCTGAGGTCAGGAGACACCTCACCAGGAAGGGGCTTCTACCCAGTGTCCTCAGTGCTGCCCCTGGGGCTTTGACCTAGGTCTGTGCCACTCTACAGTGGCAGGCCCTTT

This mutation is a 2235 bp deletion beginning at Chromosome 10 position 79,964,652 bp and ending after 79,966,886 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44530 TAC CCA GGT CAC TGG CAT C Wild type Forward A
44531 TGA GCC CGT TAG GCT GAC A Common A
44532 CTG ACC CTG TGG CAA ACT G Mutant Forward A
44533 Fluorophore-1 CAT CCG TCC CCA CAA CAT A Quencher-1 WT Probe
44534 Fluorophore-2 ACA TGG CCT TAG CCT CCC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44530 0.40 uM
44531 0.40 uM
44532 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.