Protocol 43932: Probe Assay - Serpinb13<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

>chr1:106995614+106995732 119bp ATGGCTTGCATCTGACTTGG TCTGGTTCAGAAGGATCTGACT

Mut= 86 bp

Wt= 119 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case):

CAGAAATATCCAAGTTTTAGTGGAGAAGGATTATCTGGGGAATTATTTACCCTTGTCAAATATTTAGCATCTATAAATAGATAAAACAAATGCAGGAAACACTTAGTATGCAGTTACATTTCAGACCCAAAGTGCAAAATGGATCAGAAAAGACAGATCTGTGGTATTGGGGCTGGTGAAGAGAGCGGCTTTATGCAGCTGAGATTGGTTATGGCTTGCATCTGACTTGGCAGAAAGTCAAATGGCAGTGGGGACCAATGgacatctctattcttccttcagatttgttccccagaggaaggattgtagtcagatccttctgaaccagaatctagacttgcctctgcccagataacccactgaggtttttgtcttgattttgaagatcgagaagagagaagaaatacatcatcaactccaaatgctgttgactgaaataagcaaattttctaacgattatgacctgatcataagcaacaggctgttcggagaaaaaacatacctcttccttcaagtaagttttgcagtgtttgccacatctatatgtggtaccaagaaatggccagtatccctgttaggactgagttctggggctatgctgccatgttggctgcttgccatgggactatttcactagtcattctagtctgctttccagacaagcagttcctttcttagtcttctgagtctgtatctgtttttttctttaattgtatctgcctgagcatgcatggaagaggtaatgagaaacatttttctcggatctaaaagtccaggttttgagattagttctcctaacgaacagacacatttagttaaaaagcttttgtgcttaataagtaaatccttccatctctttctatttctttcctaaagaaatacatagattatgttgaaaaatattaccatgcatctctggagcctgttgattttgtaaatgctgcagacgaaagtcgaaagaagattaattcctgggttgaaagccaaacaaatggtagagtatgaattgggttttcaccaagaaacaccttttgaacattggctgtgaccaaagccttgggcttgatgcaacacagatgccagatggttgagctttgggagaagctcgagatgggtttggcagaattgaacagtcaagcatcagcataaagctcatctagggtgccgcagagggtagaatggcctctgCATTCGTGGAGAGATGCAACAGTGGGGAATTGTTCCCACAGGCTGTGGCTCACACTACCTTTGCAGTCTTAGGGATGGTGCCATGGTTACCTTTAGCTTATTCACTGTACCACAGACTTTCTTTCTTTCTGAGTCTGGGGCCCTCAGTGCAAATGGCATAGAGGGTTTCCACTTTCTTTATTAAGAGAAGTTCATACAACTCTTGTGCCAGGACCCATCTGCTGCCTACAGCTAGGGTCACTTGGGTCTCTTTCACACTTGGGAATCT

 

This mutation is a 928 bp deletion beginning at Chromosome 1 position 106,995,664 bp and ending after 106,996,591 bp (GRCm38/mm10). 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
64380 ATG GCT TGC ATC TGA CTT GG Common A
64381 TCT GGT TCA GAA GGA TCT GAC T Wild type Reverse A
64382 GGA ACA ATT CCC CAC TGT TG Mutant Reverse A
64383 Fluorophore-1 CTT CCT TCA GAT TTG TTC CCC A Quencher-1 WT Probe
64384 Fluorophore-2 ACC AAT GCA TTC GTG GAG AGA T Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
64380 0.40 uM
64381 0.40 uM
64382 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.