Protocol 36230: Probe Assay - Scfd2<em1(IMPC)J>-Alternate 1
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

>chr5:74482088-74482191 104bp AAGCAGCCAGCAGAGAGAAC TGCAGCTTTGCTTCTTTTGA

Mutant=  120 bp

Wild Type = 104 bp

Sequence

Wt Sequence deletions in lower case t insertion in <t>:

 CATACTTTCATGGAACAATGTAGCCTTTCTTCTCCCCTTTATCCTGAGTTTGTTGTCCCCTTAATTCCTTAATATCCTGCCCATTAAATGAATTAGACACCCTGTATCAAGTTAATACTTCC<t>cagtggcaatgatggtattagtgctcctatttcccagtaggaatttactctaagctgtatgtgtttccactgcagtgaggcctcagccaaggccctgtgggaagccttactgaactccaagcacaaagaggcagtgatggaagttcggagacatctagtggaagcagccagcagagagaacctgcccatcaagatgagtatgggtaagcccctgcctctgCGGAGGCAGCCCAGAAATCCTGTTTTCAAAAGAAGCAAAGCTGCAGCTTTGATATTGCCCTGGTGTCTGTCTATTCATTCCG

This mutation is a 220 bp deletion beginning at Chromosome 5 position 74,482,133 bp and ending after 74,482,352 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46663 AAG CAG CCA GCA GAG AGA AC Wild type Forward A
46664 TGC AGC TTT GCT TCT TTT GA Common A
46665 GTT TGT TGT CCC CTT AAT TCC Mutant Forward A
46666 Fluorophore-1 CCC ATC AAG ATG AGT ATG GGT AA Quencher-1 WT Probe
48173 Fluorophore-2 TAA TAC TTC CTC GGA GGC AGC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46663 0.40 uM
46664 0.40 uM
46665 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.