Protocol 35044: Probe Assay - Arl5a<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

>chr2:52416793-52416912 120bp CTGCCTGGAGAGATTGTTGG GCCTTCCTAGAGCTCAAAAGATAC

Mutant=  137 bp

Wild Type = 120 bp

Sequence

Wt sequence with deletion in lower case

TTCAGTAACTTAAGTCATTTGTAACTGTTGTGACTACATTTGAGGATCCTTTGCTGAGCATAGACTTCCTTAGGCCTAGAAAGAGAAGGTGCTTCTGACTGCCTGGAGAGATTGTTGGAAAGATGAGAAGTGGCCGTGTAGaatgagaagaggcaaagaaagtgagcttaacaacacagtccttgcttgatggagtatcttttgagctctaggaaggctaaagagtgttgtgtgctgacttataccacagaaacaaagaagaataaagtgtgggatctctgtgtgtggtaactctctgctggtttttatttcagagcacaaagttatcattgttgggctggacaatgctgggaagacgaccatcctttaccagttgtaagtattaactgcttaaagtttaattttgttgtttctagttattctctgtgcttttgtggtatttgatggatgaaatcaatgaaaagttatgtggatgcaagcagactaaccgtaagccagaagccagggactgtgtgctgagctagaaattcgcacgataccaggcatgctcattgttttgctttgatcctcttgtttttagaaaacaaggtatctgtttatacactttgcctgaatgatctgtaatattctaaaaccattatttatttgagctcttttatatctttagctgaatcattgtgtgagcgcaaaaacaagggcaattgggtctgagttagaagccatgttgacgtaccttgatgtaagcattggtgagatgaactagcttcctcactaattttgtaaacttttcccctcctagttccatgaatgaagtcgtacacacatctcctacaataggaagcaatgtggaagagattgtggttaataatacccgtttcctgatgtgggatattggtggccaagaatcacttcgtccttcctggaacacgtactatactaacacagaggtaacattcctaatgctagtgcttgcatgagctcctgagtcctggaacacgtactatactaacacagaggtaacattcctaatgctagtgcttgcatgagctcctgagtccttgagaagcattgggttcgagtcattacagtctttaaaaatggagcgtgagtacagtagtcttctattccagtcttgactcagttaacgttgagtctctgtagGGAGAGTTGGTGGGTGATCAGTCGGTTGTTTGTCCAGTAGTTACTCTTAAATGACTTGCATTTCATGATTTCTGATTGCATTTCCTTCTTGATGTTTATATGTGTCTGAGTGGGAGAAATCCCAGCCAAGTAACGGACTCTTATGTGGTTTGAGTCCTAACCAGTTTTTGCCGTGTATTGTCTTTTATGTAGTGTAGTTGCAGCTGAAAAATCTTTTAATCATAAAGCATTTTACACATAATCATATGGTTTTATTTACATGTTACATTC

A 1020 bp deletion beginning at Chromosome 2 position 52,415,850 bp and ending after 52,416,869 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
45112 CTG CCT GGA GAG ATT GTT GG Common A
45113 GCC TTC CTA GAG CTC AAA AGA TAC Wild type Reverse A
45114 CAT CAA GAA GGA AAT GCA ATC A Mutant Reverse A
45115 Fluorophore-1 AGC TTA ACA ACA CAG TCC TTG CTT Quencher-1 WT Probe
45116 Fluorophore-2 TCG GTT GTT TGT CCA GTA GTT ACT C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
45112 0.40 uM
45113 0.40 uM
45114 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.