Protocol 32409: Probe Assay - Samd11<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

>chr4:156250527-156250614 88bp TCCTCCTCCCACTCTGTTCA CCCTGATAAGCTCCCGTAGG

Mut= 86 bp

Wt= 88 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 

CCTTTCTTCTCCAGTTTCGATGACCTACTTATTCACATTCCTTTTCAAGTTCACTTGCCTGGCTACAGCATGTAGGTGTTAAAGCAAAGTGCCTTCCCTGCTGATCCTCTAGCCTGTGACACAAAAGCAGGCTCAGGACATGTGAGAGTCTCCTCCTCCCACTCTGTTCATACCTTCACCAGGCCAATgccctgctcaccaagatggaaaggggaaatcctacgggagcttatcaggggggcccgctatcacctcccttctcagatctgcttagggtacgccaggaagtagcaactgcaacactgaggggtcccagtggcttggaagttcacctgccctcgtctacagcagaccatcgacggaagcaaggcctagttcagcgccgggaaggggcggttccagctgctgccacatctttctctgaaaggtattaagacgtgcttcagcaaggccccacatgctatgcttgttacttgagaaggccaattctttctttcccttgagtcttccatcacatatatgcataatgcattcaccacgttcaaggtccactgcaggaagctcctaaaactactgcaaagactcaatttggggggcaaaatatgcagaaatgcccactccctggcaagaatgccaaatgcaattttcttgtcaagcaactctgagtattccagcctagtagagctgccccgagagcagaggccctcCCCTCACAGGTGGTGGTGGTGGGATGTTGAGGAAGGCAAGGAGAAAGCAACCACAGAGGATTCAGAGTGAAGGACATACGCCCCCTCCATCCCTACCCTGCTATAAACTCCAGAGACATCATGCTCACAGTAGTCTCCGCGGCAGACGAGTTAAGCCAAGGAGAGCATTAGGCAG

This mutation is a 519 bp deletion beginning at Chromosome 4 position 156,250,058 bp and ending after 156,250,576 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
39037 TCC TCC TCC CAC TCT GTT CA Common A
39038 CCC TGA TAA GCT CCC GTA GG Wild type Reverse A
39039 GCT TTC TCC TTG CCT TCC TC Mutant Reverse A
39040 Fluorophore-1 CCC TGC TCA CCA AGA TGG Quencher-1 WT Probe
39041 Fluorophore-2 CCA ATC CCT CAC AGG TGG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
39037 0.40 uM
39038 0.40 uM
39039 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.