Protocol 31950: Probe Assay - Svopl<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=  97 bp

Wild Type = 106 bp

>chr6:38041123-38041228 106bp CCATGGTCCTCAAGGGTTC ATGGTCTCCACCGCATCCT

Sequence

Wt Sequence (deleted region in  lower case)

 

GACTATTTGCCTAACCTGCACAAAGCCCTACATTTGATTCTCAGTACCACATAAACTGGGTACAGAGGTACCTGTCTGTAATCCCGTGTGAGAGGTAGAAGCCTGAGAGTCAGGAGTTCAAGGTCATCCTCAGCTACGTAGGCGTTTGAAGTCAGTCTAGGCTACACAAGACGGAATAAGAAATAATAATGTTGCATATACCTGTAGAGGTGGGCCATGGTCCTCAAGGGTTCCCCTGCCCCCACACCAAGTGgttctcatactctgccttccttgtcagagaccaagacattcacggtggaggatgcggtggagaccatcggcttcgggcgcttccacattgcactcttcctaatcatgggcagcaccggggtgagcacacttggagaagctctgtggctgtgattctgtcctcactgtttagcaccaagttcctgctgacccccagcactggactgcccattcccgggagtaggaatcctgtggaaattatttcatcagcctttgcagtgggttggctgggaacgtgtctgttctggtaattcaagactccatcttagaggtttacactgagctcttgggaagaagggaaaggactgtggggttactccacgtggagtagctgcaagCTCTGAGCATGTGTGCATTGGTAACACCTGGAGCACCTGCTGTCGGGAAGACTCTAGATCCCCTAAGAGACTCAGTCATTTCCTTCATATCAGTGGTATTTGTTTCTCCAAAATCCTTGGTGTTGTGTGTTAGGCATGGTGGGTGCATCTGTAGTCACTGTATTTGGCAGATAGCTATAAGAGAATCAGGAGTTTAAGGTAATCTTCTGCTAGGTGTTGAGTTCGAGGCCAGCTAGGGATATGTGAGATCTAACTTTCCTCCAGATGTACACAGT

376 bp deletion beginning at Chromosome 6 negative strand position 38041189 bp and ending after 38040814 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
38035 CCA TGG TCC TCA AGG GTT C Common A
38036 TCT AGA GTC TTC CCG ACA GCA Mutant Reverse A
38037 ATG GTC TCC ACC GCA TCC T Wild type Reverse A
38038 Fluorophore-1 TGA GCA TGT GTG CAT TGG T Quencher-1 MUT Probe
38039 Fluorophore-2 TCT GCC TTC CTT GTC AGA GAC Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
38035 0.40 uM
38036 0.40 uM
38037 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.