Protocol 33531: Probe Assay - Rusc2<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

>chr4:43424380+43424477 98bp GTGTCTGCTCCTTGGGATG CATGGCCTCTGAAGTGGAG

Mut= 99 bp

Wt= 98 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 

ACTCTAATTGAAGGCTGTAGTTGGAGCGACTCTGGGCAAGCATGTGGGTGACAGAAGACAAGAATTGGCAGGAGCATGCTGGTCAGTAGGTTAGTGACCAGTAGTCCAGGAGCTActacagggtaaataaccaagtgggccaaggggcaggagcttggccaagaaggcggagttgcttgtagagccaaacaaaacgcaaaggtcagagagggaaagctgtccatgaagagagcagccaacccccggaaaaggaaagacccacgagaaccgtcggtggggaggggcaacttcctcttccccactcaggtgtccgcctccaccaggtgggctagcatctccccaaggggacctgtgacagccacttctccctcacagggctggtaaaagctgtcaacaccgctgtggaccttattgtggcccattttggcacgagccgggatcctggcgtgaaggtatgctgacactatcaagagctgagctatgccctgcagactcggccgtcacctccctctgctctcggggacggaattcggggacagaaatggccgggggacatccaagagggtctgactaagccagactaaggtgaggcttctcacggttgccctatgtatccgcaggcaaagctgggcaacagttctgtgagccccaatgtcggccacctggttctgaagtacttgtgccctgcagtccaggccgtgctcgaggacggactcaaggccttcgtactggatgtgatcatcggccaacgtaaaaacatgccttggagtgtggtcgaggcttccacacagctaggtaggtactagattgcagccctggactccggactgaatttggggttccagaagttagaaatgtgatctccaaacctgccttctccccaggcccatccaccaaggtcttgcatggcctctacaacaaagtcagccagttcccagaactcaccagtcacaccatgcgcttcaacgccttcatcctcggcctgctcaagtgagtgcagaatggcaagatgtaagcagccagccccaccatagctttgcttcctctcaggggctggctgcctggaaggagaccggaggggtgtctgctccttgggatgcctttaccccaccgaggtgcagCCTCTCGGCAGGCGCTGACACCTCCCACCATCAGGCCCCTCCACTTCAGAGGCCATGGCCTGGCCTGTTTAGAAAGAAGCTGGGTTTCTCAGTGTTCGGTGTCTGCAAACGGTGATGTCTCA

This mutation is a 1006 bp deletion beginning at Chromosome 4 position 43,423,415 bp and ending after 43,424,420 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
41624 GTG TCT GCT CCT TGG GAT G Wild type Forward A
41625 CAT GGC CTC TGA AGT GGA G Common A
41626 GCA TGC TGG TCA GTA GGT TAG Mutant Forward A
41627 Fluorophore-1 TTA CCC CAC CGA GGT GC Quencher-1 WT Probe
41628 Fluorophore-2 AGC TAC CTC TCG GCA GGC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
41624 0.40 uM
41625 0.40 uM
41626 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.