Protocol 31952: Probe Assay - Uck2<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

Mutant=  96 bp

Wild Type = 115 bp

>chr1:167237034-167237148 115bp GACCAGCAAGAAGGGAGATG GACTCCCAACAACGAGAAGC

Sequence

Wt Sequence (deleted region in lower case)

 

CATTGCATAGCCCTTGCTCCGGCTCTCGCTCCCACTGATGAGGCTGTGTAGGCGCTGAGTGCCCCGTGCTTACGTAGTCACTTTTTGCTCTGTCAGCATACCATCAGTTCCACTTGAAGATGGAGCGATACTTGGCATTCCGTTTTCATTCACCAGCTTGCAGAGAGGAGGCAGGGCCACTCTCCACACCTTACAGCATGGAGCTCTCAGAGCAGCAGGCGCTGAGGAGGAGGAAGGGACCCTGGGCTCCTTAGACCTTCCTTTCAGCAGGCTGGTCTTAGAGGGGTTTTATTTTTCAAGATCCCAAACCCATCCCCACCCCCACctcggcactgtcttaccgttttagttgactgtatattactagagctcagggaggcgccagcctcgagatgtgcacttcctgtgctcccacacacctctgactcctggctgtcgtcttctctcctcacagtcttccgtttgtgctaagatcgtccagcttttggggcagaatgaggtggattaccaccagaagcaggtggtgatcctgagccaggatagcttctaccgagtcctcacctcagagcagaaggccaaagccctcaagggccaattcaactttgatcacccgggtgagttgggagttgagagatgtgtgtgttgaaggtggatcgggtggtggctgtgtccacgaggcttagcttctcccagtctgttcctcctgtccacacataccataggggcctaatgaccagcttgcagggttatcatgtgcttagaggaaagaatgggttttgggatattttgcagagagctgattgaatcgaatgaatgccagccatgtaagcttgggtagagatgggtcagcccccggagcaaacacaccactggcctttctgattgatgtgtagcagcctgtaccatctccacccacccctcaacccccaccctgccccatgtggtctgcttggtgaccagcaagaagggagatgggagtcttaacgtggacccctacgtgaattctgctgcgagcttagatcagcacagtgctcCTTCAGGTCGTCTGGGCTTCTCGTTGTTGGGAGTCAGCCCTGCTTGGCATGGGACTGCAGTTCTTCCTAGTCCCGGTCTACTTTCCACTTAGCTTGAGCCAATCCCAGGGCCCCCAGCTGACCTTCGAGGAGAGATGGGAGGGGCCATGGCAGCACCAAGTGTAGGCTGTCAGCAGGTAACAAACAGCTCAGTTTGCCAGAACTCATTTAACACACCCTGTTTTAAGATCCAGGGAGTGTTTATATTCTGTCACGGGTGGACACTGGCTTTGGAGAGGCAACATACGGGGTGTGAAGTTTGTTCTGATTTTAGGACAGGTTA

709 bp deletion beginning at Chromosome 1 negative strand position 167,237,777 bp and ending after 167,237,069 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
38042 CAG CAG GCT GGT CTT AGA GG Mutant Forward A
38043 GAC TCC CAA CAA CGA GAA GC Common A
38044 GAC CAG CAA GAA GGG AGA TG Wild type Forward A
38045 Fluorophore-1 AAG ATC CCA AAC CCA TCC C Quencher-1 MUT Probe
38046 Fluorophore-2 ACG TGG ACC CCT ACG TGA Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
38042 0.40 uM
38043 0.40 uM
38044 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.