Protocol 37451: Probe Assay - Slc17a1<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

>chr13:23874590+23874719 130bp CAGTTCCAGGGTTCTGTTCC GGTGACCCTGTGTTGTTCAC

Mut = 150 bp

Wt = 130 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

ACTATCTTGAGGAAAAAAATTTCTCAGTGTGCAACTGTCAACCAGAGTAGTTATATCAGTTTGAAGATGAAAGTATTTTCTTTGAGAGAAAGGgacttacttccaaagggaacccaaggtattcatcaaggatccattcaaaatagacccacacccaaacatttggaactgaattaactttctgggacattctaattggaacaatttcttaaagtaataaattattaaatcaatatcgcattttcccattaaatgataatttctagaaggatgttctgcaacaaaattttaacttactatctagtatatctagggttgcaaatacctgaatttttacttcaactgtgttgctaattgagattttctttttcaacttctcagttccagggttctgttccttccggtatggactggctatccttctgcacttctgtaacattgccatcatggcccagagagtgtgcctgaacctcaccatggtagccatggtgaacaacacagggtcaccccatttatctaacgaatctgtagtggagatgttggacaatgtaaaggtatagtgaaagtgtaaagtgcaagtttaaaacttaaactcgtgagctaggttctacagagGTCAGTTATTATTGGCAAGATAATAGGAATAATTAGAAAATGAAGCTTAAGTTTAAATGTGGACGACAGCACTCACTAGACTGGAGTCTCAGAATGAACAAAAAGAAAACAGG

This mutation is a 522 bp deletion beginning at Chromosome 13 position 23,874,305 bp and ending after 23,874,826 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
51356 CAG TTC CAG GGT TCT GTT CC Wild type Forward A
51357 GGT GAC CCT GTG TTG TTC AC Wild type Reverse A
51358 TGT GCA ACT GTC AAC CAG AGT Mutant Forward A
51359 CCA GTC TAG TGA GTG CTG TCG TC Mutant Reverse A
51360 Fluorophore-1 CCA TCA TGG CCC AGA GAG T Quencher-1 WT Probe
51361 Fluorophore-2 AAG GGT CAG TTA TTA TTG GCA AGA TAA TAG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
51356 0.40 uM
51357 0.40 uM
51358 0.40 uM
51359 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.