Protocol 37557: Probe Assay - Smarce1<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

 


>chr11:99219394-99219500 107bp GTTCTGTCCCGACATCCTGT GAAAGTCAAAGGGGCTGGT

 

Mut = 156 bp

Wt = 107 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case):

 TAAGTACAGTAAAAATATTGATCATAGTTAATACATATGTGTTTGGTAGTATGACAAATTAACTACCTTGAATTGACTTGAACAGTTAGAGACTTAGAGTTCATTTAATTCTCTACGAGTGTGAGCAGTGATTAGtggtgcccttagcttaatcagtttcaaatatttatgagcaactgacagtggtgagtaagatccagtgggataaggagggcatggggtaagccagctgttggcagttttgaaacaggctgattgctacaaaggagtttgatactgtcttctttgctttttcttgtatttaaacgtttccatattagagtgtttttaaatgactatatacttaggcactaatattgaattttcaactattgactttttaaggtctgggaccaagtaaaggcttccaaccctgacctaaagttgtgggagattggcaagattattggtggcatgtggcgagatctcactgatgaagagaagcaagaatatttaaacgaatacgaagcagaaaaggtaggaggactgccttggcttattatttataaaagaaaccaagcgtgaccagatggctgagccacactgcagttttgtccaccaagcttcactcatctgagtggcacattctttgttctgtcccgacatcctgttttacctctccctctgatgttcagagggacagtgcaggtggcctgtggagtagaatggacattataaaaccagcccctttgactttcagatagagtacaatgagtctatgaaggcctaccataattcccctgcgtaccttgcctatattaatgcaaaaagtcgtgcggaagctgcattagaggaagaaagtcgacagagacagtctcgcatggagaaaggagaaccttacatgagcattcagcctgctgaggatccagacggtaaggtggaaaacggggctttgtatatgtattctctctgaattagaaaagaaatttagttcaaggcatttttattcttttaaatggaaaagcataattttaagtctttttgttgtagtaagattttaagtagtaagatttttgcgtgtgactttctcatggaaaaaatactccttacagggtGGAAAATTATCAAAGAAAAAAAGCTGTATTTATTACTTATCAAAGTCTTTATACTCAGAATAGAGGATTTGTTTGATTTTCGAGACAGGGCCTCACTGTGTTGCCCA

This mutation is a 955 bp deletion beginning at Chromosome 11 position 99,219,039 bp and ending after 99,219,993 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
50050 GTT CTG TCC CGA CAT CCT GT Wild type Forward A
50051 GAA AGT CAA AGG GGC TGG T Wild type Reverse A
50053 GTC TCG AAA ATC AAA CAA ATC C Mutant Reverse A
50054 Fluorophore-1 AGT GCA GGT GGC CTG TGG Quencher-1 WT Probe
51669 CCT TGA ATT GAC TTG AAC AGT TAG Mutant Forward A
51670 Fluorophore-2 AGT TCA TTT AAT TCT CTA CGA GTG TGA GCA G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
50050 0.40 uM
50051 0.40 uM
50053 0.40 uM
51669 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.