Protocol 34824: Probe Assay - Vkorc1l1<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

>chr5:129978374+129978501 128bp GCGCAAATCAGTCATGGA CCAGTAACCCTATATTTGAGATTCC

Mut= 130 bp

Wt= 128 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 GGCTTTCCCTTGCAAATGCTAATACTGTCAGTAAAGAAACAGGGTGCCTACCTTAGGAAAAGAAAGTTTAGAGAGTCTTAGGTGGAAATACAGAGCTTTTAGGGATATTTGTATTAAACCATCAATATAATTCAGACACAGTACAGAAATCTAATGTTTGGCCTTACCCTtgcgcaaatcagtcatggaaaaaaagatatttaagtgttttgcaaacatagttgattgtggaattggataatattctcaaataattttacatttaaaaaattagggaatctcaaatatagggttactggttttccttattttacagatggggtcgaggatttggtcttttgggttccatttttggaaaagatggtgtattaaaccagccaaacagtgtctttggacttatattttatatactacagttattacttggtaagtattgatacagagatacagaaataagcttgttatacccattcagtgcttgtgtcttcaccctagTGTGTCTTCATAGTAAACGTACACGCTAGTAATAGAGAACCACGAGAGTGAAGTGTGAGTCACTGCTGCTTATCATGTGTTGACCTTGGTATCAGTGGACGTTTAGTCATGGCTAGTAGTGTGTCATGATGTCCAGAAACCCAGCCACATTCATGGAAATT

This mutation is a 325 bp deletion beginning at Chromosome 5 position 129,978,373 bp and ending after 129,978,697 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44580 GCG CAA ATC AGT CAT GGA Wild type Forward A
44581 CCA GTA ACC CTA TAT TTG AGA TTC C Wild type Reverse A
44582 GTG GAA ATA CAG AGC TTT TAG GG Mutant Forward A
44583 GGT TCT CTA TTA CTA GCG TGT ACG TTT Mutant Reverse A
44585 Fluorophore-1 AAA CAT AGT TGA TTG TGG AAT TGG A Quencher-1 WT Probe
44587 Fluorophore-2 CTA ATG TTT GGC CTT ACC CTT GT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44580 0.40 uM
44581 0.40 uM
44582 0.40 uM
44583 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.