Protocol 47812: Probe Assay - Olfml2a<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=  108 bp

Wild Type = 118 bp

>chr2:38950922+38951039 118bp GACAGGAGATGGGAATGAAT AACGTTGTTCCCAGAGTAAA

Sequence

Wt Sequence (deletion in lower case):

CCCATAAACCAAGACCAGTGAGCGAGAGACAAGGCTAGGAGGTCTTCCAAAGAGCCAACAAAACAGGTGGACAGGAGATGGGAATGAATATGTAACCTACCggattgggtctcaagcctgcagagaaaatctgtatggagaaacagaatccaacaactatggatgattttactctgggaacaacgttacagacactgcgactacctccagctctcacacactgagctactgtgactggcagcccctcattttactgtcacccaaatctgacacttctattcttcccaggacacagctggagggcaaggcagagatctcaacaaatatggcagcatacagaagagcttctcagacaagggcctggcaaagcctcccaaggagaagctgctgaaagtggagaagctgaggaaggagagcatcaagggcagaataccccagcccacagccaggccacgtgcgctggctcagcagcaggcagtgatccgaggcttcacctactacaaggcaggcagacaggaggccagacaggaggccagacaggaggcacccaaggctgccgctggtgagtggaagggagccgggcctggttgacggatggctgcttccagagatccacctgccttcgaagcagccacaaagtcacagatctccgcatcactgtttagttttctagaatcctttgctcaaccatacACTGGGAAGAGGCCCACAGGCAGGGTTGCTAATGGGGAGAGAATAGAGGCCTCTCTTTCAGTACTTCAAGCCTCACCTTTTCTGCACTTCCTTTTATTCAACGACCTGCTGCAGGTCAGCTACAGACCAAGTGAC

 

This mutation is a 592 bp deletion of region Chr2:38,950,954-38,951,545 (GRCm38/mm10)

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
70979 GAC AGG AGA TGG GAA TGA AT Common A
70980 AAC GTT GTT CCC AGA GTA AA Wild type Reverse A
70981 GTG AGG CTT GAA GTA CTG AA Mutant Reverse A
70982 Fluorophore-1 TCT CAA GCC TGC AGA GAA A Quencher-1 WT Probe
70983 Fluorophore-2 ACC TAC CAC TGG GAA GAG GC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
70979 0.40 uM
70980 0.40 uM
70981 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.