Protocol 34707: Probe Assay - Ubtd1<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

>chr19:42031763+42031872 110bp GTCCCAGTGCCCTAAATACA CACAGGTTCAGCATCTTGGA

Mutant=  100 bp

Wild Type = 110 bp

Sequence

Wt sequence with deletion in lower case

CTCTTTCCTGCTATTCAGAAAGTGTCCCAGTGCCCTAAATACATAGGGACTGGTAGAGTGAAGTCCCTGATAggccagggccagagagggggtcagttacagcttaagtggcttccaagatgctgaacctgtgcacctgtcttacctaggtcgcaatgagcccttgaagaaggagcggctcaagtggaagagtgactatcccatgactgatgggcagctgcggagcaaacgagatgagttctgggacacagcacctgcctttgagggccgcaaggagatctgggatgccctcaaggctgctgcctatgctgctgaagctaatgaccatgagctggctcaggccatcctggatggagccagcatcaccctgcctcatggtaagcaggcagggcctgggctctgtccttacctggactggggctggaggtttcattagcgcagaggtggaaagcaagtttctcatgtgagaagtttctgttttagctggtggagatgaaggagtatgcctatatgtgtgactgtcttcttcgtttaaatgactctttatggcatgaacaaagaagcacaaggcacagaaccaagcgtagtggcacatgcctgtggtcatgagaggtagacacagggggagctgaagttcaagttcaagaccagcctagattacatgagactgtcagaaggaggaaaggaaggagggagggatggacagaatccccctttgcctagtgagaagacaggaagacaccaataaaacctacaagtttaaaaggcactcagggcctgagagcatttgaagcactgatctggaaccttctgctgcattgccgcatttctgttcctagcaccctcattagctgtgaagggaaagtgacagtgaccttcagtcacagacctgggtctttttttgtttgtttttgttttgtttttcaagatagggcttctctgtatagccctggctgtcctggaactcactctgtagaccaggctgccctcgaactcagaaatccacctgcctctgcctccggagtgctgggatcaaaggcgtgcgccaccatgcctggcttcagacctgggtcttgactgtgttcctttctcagcttgtgacttttcctagccatatgtcccagagcagggcaagtctcaggttagagcctgagtctcttgcaatcacaaaggattaatcgtgatgtgcatagtaccacctagtctagtaccagcacttacaaggatactgaagactaaataaatgtgtttccacctgcattatatcataccagtaaggagacagggacccaacccaacacttgcatgtgcattcagccaccacaatgttgaataaacacataagaggtagcatcctgtggctgaggagccagaacctgatgaccaggaggttagccccagtctactgactatggggaagggctagggacagtgaggtagctgtggtgagtgaattaacctgcttacactgttgtagcagaggaccacaggcaggctccttaaacagtagaagtttgtttctcacaatttgagagactggaaagtccaaggtcaaaggcccagctgtttagtcctgatgatgcttcttggtttttttgctgtttttacgtggtaggcgtgcagaagagaggactgggtgggttctctgtttttatttggtaggcatgcaggaagagaggactgctcgggttctctggggcttttccttgaaaggaagctggtcctgtcatttacccgtgtaatcttattccttccaaatatgccttaacatgggagtttgggggacacagcattcagtgtgtgacatcaggccccccagactacactgatcaccctgcctttcccacaggaactctttgtgaatgctatgacgagctgggtaaccgctatcaactgcccatctattgcctgtccccacctgtgaacttgctgctggagcacacggaggaggagagcctggagcctcccgagcccacccctagtgtgcgccgtgaattcccactgaaagtgcgcctctccacaggcaaggacgtgaggctcaatgccagcctgcctgacacggtggggcaactcaagaggcagctgcacagccaggagggcattgagccatcctggcagagatggttcttctctgggaagctgcttacagaccgcacaaggctccaggaaacaaagattcaaaaagacttcgtcatccaggtcatcatcaaccagcccccaccaccccaggactgacggactgacggactgacggcaccaactcctaggaaaagaggccatggggggccctgcaggctgtggggcccagtcctgccgccttccagtgctgctgtcttcttcaggggcgcctcccaccgccctgctgtatggctcttagtgagctgcgaagggacttgccctctggggcactggatccaccagtgctcagcacccagggagcagtgctgcctgccacacacaggccttgccaaccttggagaaaaggcacatggcactctccaccacaggttggagccatgggggccgtcctggcgacctgtcatctggtgccacgctgtgctcactccagttttctgctcagggttggaagcaattgctgtctcccctttatccctcaccatgactctgccctgggcacagtgccagtcccctggatggggagggagcccggtgggccccagggcttaaggagcttcaggtgggcctctctgcctctccctgcacccagcacaatctgcagagatgaggcgggtggctggatagccaggcttcatggcaggggtttgcacagggctgtccccaactgtaacccaggcagggtggggtccgaagcctggtcatagcccagcggggccctgtgtacagacacatctgcattttatcaataaagcctttgctcctctcttgcctagcacatgacttcctgtttcacagcacgccagccagccggagcccagagcttctgctggcttggctgcagatgggcacagcagagcatccaactgatgctcctcctgGGGGCCCTAGTCTCATCCAACCTCGCTGTGTTCTGTGGGTCCTGAAAAAGGCCACTTCACCTCTGAACACAGTTTGCT

 

A 2945 bp deletion beginning at Chromosome 19 position 42,031,812 bp and ending after 42,034,756 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44266 GTC CCA GTG CCC TAA ATA CA Common A
44267 CAC AGG TTC AGC ATC TTG GA Wild type Reverse A
44268 CCT TTT TCA GGA CCC ACA GA Mutant Reverse A
44269 Fluorophore-1 ATA GGC CAG GGC CAG AGA G Quencher-1 WT Probe
44270 Fluorophore-2 CTG ATA GGG GCC CTA GTC TCA T Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44266 0.40 uM
44267 0.40 uM
44268 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.