Protocol 37240: Probe Assay - Smarca2<em1(IMPC)J>Alternate 1
Version 2.0

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

 

>chr19:26646876+26646984 109bp AGTTGGGTATCAGAAGTTGACTG GGTGGAGACCTGCATTATGA

 

Mut = 95 bp

Wt = 109 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case):

 TACATGCACTATGGTATGTACATTTCCCCCCTCTAATAAATAAATAATTGCAGTAATTTTTTTTCTTTTAAAAGAAAAGAAAACTTAGTTGGGTATCAGAAGTTGACTGTCAGTATATGGAatataatttactatgaacccaaacctaaaacattgctgatcatagaggaagaaatcataatgcaggtctccacccaccccacccctgtcttctttcttttttctttctagacttcaagctcgcatcgctcataggatacaagaactggaaagtctgcctggttccttgccaccagatttacgcaccaaagcaaccgtggaactgaaagcacttcgcttactcaacttccaacgtcaggtaatacctttttcccccgtgatgtaggaaATCAATGTGATTGTTGCTAAGGTGTGACCTGGGAGCTTCCTCAAGTCTACCTGATCTTGTTATAAAGCTTAGTGAGATAGTGCGTCTTCAGACTGACAGCTCACGGCTGAGAGTGAGGAGTATGAACCGCTCGTT

This mutation is a 267 bp deletion beginning at Chromosome 19 position 26,646,911 bp and ending after 26,647,177 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49710 ACA AGA TCA GGT AGA CTT GAG GAA G Mutant Reverse A
49712 AGT TGG GTA TCA GAA GTT GAC TG Common A
49714 Fluorophore-1 ATG GAA TCA ATG TGA TTG TTG CTA AG Quencher-1 MUT Probe
50858 GGT GGA GAC CTG CAT TAT GA Wild type Reverse A
50859 Fluorophore-2 CCA AAC CTA AAA CAT TGC TGA TCA TAG A Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
49710 0.40 uM
49712 0.40 uM
50858 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.