Protocol 35631: Probe Assay - Lpcat2<em1(IMPC)J>Alternate1
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

>chr8:92865160+92865264 105bp CATGAAATGTCTCCAATAGCTTC CTAGTCCTTTGATCAGGCACA

Mut= 96 bp

Wt= 105 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 GTGACACACCTACTCCAACCAGGTCACACCTATTCCAACAAGGCCACACCTCCAAATGGTGCCACTCCCTGGTCCAAGAATATACAAACCATGATAAATGTCCTATCTTGTGTACATATTTGTTTGTGTGTGTTTATATCTCAGTCTCATGCAAGGCATTTAATTAATTCACCcttgagaacatgcaggaaaccaaaaactttcctgttacttacttttttttttttttttttttttttttttttggtcaactttggtttgcagatgtttcttcttggaattattctgctcccagtccgtgctttactggttggaataattttgctgctcgcatggccatttgctgtcatttcaacagcatgctgtcctgaaaagctgacccatccaataagcaattggaggaggtaagtggtaatcatgtccaaatattacacaaatcaaaacttttaaagtttctccaatgaccagtttttgtgaaggatattcatctttggttctaaagagcccctgggatatgttttaacatgaaatgtctccaatagcttcatgcattagaggctttattgacagccaatgggCTTTTGGGGAGTGACTGTATCTTGAGGGCTGTGCCTGATCAAAGGACTAGTGCCTTTATGGATTCATAATATGATGGCATTATAAGGAGGTGGTGAAAATCGGGGGGGGGTAGCCTAGTTGGAATAATTATGTCAATTACTAGGAGTATGTCCTG

This mutation is a 405 bp deletion beginning at Chromosome 8 position 92,864,810 bp and ending after 92,865,214 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
45596 CAT GAA ATG TCT CCA ATA GCT TC Wild type Forward A
45597 CTA GTC CTT TGA TCA GGC ACA Common A
45598 GTG TGT TTA TAT CTC AGT CTC ATG Mutant Forward A
45599 Fluorophore-1 CAT TAG AGG CTT TAT TGA CAG CC Quencher-1 WT Probe
46568 Fluorophore-2 AAT TAA TTC ACC CTT TTG GGG A Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
45596 0.40 uM
45597 0.40 uM
45598 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.