Protocol 35027: Probe Assay - Abhd14b<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

>chr9:106451272+106451365 94bp GGCTGTGGGCATGAATGA AACGGCAATGAATGGGAAG

Mut= 92 bp

Wt= 94 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 CTGATAGACTCCAGAGCTTCATTCTTTTCAGGACACTGTTAGAACGAACACAAACATAAAAAGAATTGTCTCATTCTCGCCATAGATAAAAAACACTGGGCTGTGGGCATGAATGAATTTCAGTCTACTACTGGGTGTTACGTGAacctgggcacagagtggaggaagggacccttcccattcattgccgttgtctctcctacgcccgactacctgcaggtcttgggcgatccaaggaagcagcagcccctgcccctattggggaaccggcccctggcagcttcctagctgctgtggtggataccttggagctgggacccccagttgtgatcagtccatcattaagtggcatgtattcactgcccttcctcgtggcccccgggtcccaactccggggctttgtaccagtggctcctatctgcacagacaaaatcaatgctgtggactacgccagtgtgaaggtaccgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgactgagatggtatctcaggagcaagtcggggtccatccctgggccttcttcatctgggagctgggtaaccaatggggtggtttctggggagaccccttttggttgacttagctgattgtttcgccaatccctgtataagactgtgtgctggggaaaccacaaaccaaggtctccatccactgcatcagcaccgcctctcctcctcaggagggcaggaagagactgaagaggtggtgaggcctgaagctaacctttagaagctgggctggtacgaccagcccccgattctgttctcctttccttcccctcagactccagctctcattgtatatggagaccaggaccccatgggctcctccagctttcaacacctgaagcagctgcccaaccacagagtgctggtcatggaaggggcagggcatccctgttacttggacaagcctgatgagtggcataaggggctgctggatttcctgcaagggctagcctgagactcagtcctgatgataggaggttacagactgcctgctctaggatctcttgctgtcttctcctggtctctggcttctgcctcacctgcaacaggtctgtctatacatctgtctgggttgctgtcatttctgtttttctcttgcagtgacagatcagatcaaggcggtaaatcaagtggaaaaagcgtcgggttcaaggggagaactctgattgtgggtaatccataagcttcctctacaggctccccgcttggttctcacttctgccttacttcactttctgatccatctctctcttcagccgctccacaggacagccattccttctgccgcccaccccccttagaccacagacagcatatgggtattgcttgtttagatccatcccacacccagacagaacctttcccttgggagcagcacaggggtcccacattcttgtgtagtttgcacaggtacatgtgcagtttcctgaaggatggagggaatctttgtaaacagagatccacttgccaggataaaaatgtggccaccgctagatttctcttacctacccgttgcaactggtcccagaccagaatggcctcctcagaaggggaggtgctcttcaaatctcccttaggggctgcatacctccccaggtaatcaagcacggatcttctcagccaagcccatttcctgccctgaggtttgggagtgggataaggagaggagaccgacttcggaggtccctgaaataaagtgatacaattagacctcacatgcttttgctgtctccggagcgccgcccctcccccttccccttgggactgccactgtgggctcctcccccaccctggcgggaccaaatggtccccctgccctttgtccactaggccagatggagggaaagggagattggattcccctccccacccacatacatccaggtgccaagagactcagctgaggggggaggtgtaggtcagtgggacctatatgagccgattactgaaagggggagaatatctgctctgagatgggggaagggcaggctcaattcccccccccccccacacaccCACCCACACACATATACACACACACACACAACCTATCCCTTTCCTAGTAAAGGAGTTAATGGCACTCTGACCCTAGCTCCAAAAGGAATTTGGCAGGAGATTGGAAGGATCACGGGACAGGTTGTTTAAGGGCCAAACGGCTCT

This mutation is a 1906 bp deletion beginning at Chromosome 9 position 106,451,319 bp and ending after 106,453,224 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
45064 GGC TGT GGG CAT GAA TGA Common A
45065 AAC GGC AAT GAA TGG GAA G Wild type Reverse A
45066 AGG AAA GGG ATA GGT TGT GTG Mutant Reverse A
45067 Fluorophore-1 ACC TGG GCA CAG AGT GGA Quencher-1 WT Probe
45068 Fluorophore-2 TGT TAC GTG ACA CCC ACA CAC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
45064 0.50 uM
45065 0.50 uM
45066 0.50 uM
Glycerol 6.50 %
Dye 1.00 X
Kapa 2G HS taq polym 0.03 U/ul
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.