Protocol 35488: Probe Assay - Uckl1<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

>chr2:181575225-181575324 100bp TGAATAATGAGGCCCTACCC AATTAATGGCAAGATGGCAGGA

Mut= 116 bp

Wt= 100 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 CATACATCTTTCTACTGGCATGTGGCCAGTATACTGCACTTTCACCTCCTGGGGCAACCAGGGGTAGGGCAGCGGTGGCTGGACTACCTAAGGAGTGTGCTGTGCTGGGGAGAGAGGCACAGTCCTTAGATCCTCCTTTTTCTTGAATAATGAGGCCCTACCCACCACAGTCcacatgggtaggattgtggctctgagatcatgcatatcctcctgtgtcctcctgccatcttgccattaattgctgtcccttgttcttgctgtgtgaacagcctgagatcttgtgccccagttaaccctatggaacaggtaaaggcaggtttcttggacacactctccccagttgagcctatattgtgcctgtcccactttggtctgaccagagaggttgaaaactgttgactgaggctaagcacattcctctttacagcagcaatgcagggaccttggacagacttctcccaccagtgggtactgggcgctcaccccggaagcgcaccaccagccagtgcaagtcagaaccacccctgcttcgcacaagcaaacgtaccatctacacagctgggcggccaccatggtacaatgaacatggtacacagtccaaggaggcctttgctattggtgagagtggtatgggaaggcaccttggggatctgtctgcatggccagcccagctgcaacagtttgcaaggtaccctttcactccacaggcttaggaggtggcagtgcatctgggaagaccactgtggccagaatgatcattgaggcactagatgtgccctgggtggtcctgttgtccatggactccttctataaggtgaggccctgctcatctacccccagagcatcttctctctgtgactgctttgcctctacaaatgtaggcctgtggtcagcagcaggcttcatcatgcccctccacccccaggttctgacgcagcagcagcaggaacaggctgcctgtaacaatttcaactttgaccaccctgatgcctttgacttcgatctcatcatttccacccttaagaagctaaaacagggcaggagtgtccaagtgcccatctatgatttcactacccatagccggaaaaaggactgggtaggcccatggtgtgggggcctacctatgggagaggttgcaggggtctgagctggacattaaaactctctgggttccttttcagaaaaccctgtatggtgcgaatgtcatcatctttgagggtatcatggcctttgctgataagacactgctggaggtgagagcttgccgggagcacccactccacaggcatccccataaatactctcttagtaatacgccgccccatcctcacagcctctatgatttgagatttctccctattgggcagttctggatgcgccccacctggtgtatcCTAGGCATTTACAGCCGTTCTTTGCCATTAGCTGACTACTACAGTGGGTGTGGCGTCTGCGGACAGTGGTTTCCTCTCCTACCTTCGGGGAGGGAGCTGTACTTCTGTCTCCCTTGCTCTTGATATTTTTCCTTGCTAAAGAACAAACAAGGACTTAAAGCCAGTGAGTTTAAGGGACTTTGC

This mutation is a 1225 bp deletion beginning at Chromosome 2 position 181,574,071 bp and ending after 181,575,295 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46202 TGA ATA ATG AGG CCC TAC CC Common A
46203 AAT TAA TGG CAA GAT GGC AGG A Wild type Reverse A
46204 CGA AGG TAG GAG AGG AAA CCA Mutant Reverse A
46205 Fluorophore-1 AGG ATT GTG GCT CTG AGA TCA T Quencher-1 WT Probe
46206 Fluorophore-2 GTC CTA GGC ATT TAC AGC CGT T Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46202 0.40 uM
46203 0.40 uM
46204 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.