Protocol 31832: Probe Assay - Serpina3k<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

Mutant=  111 bp

Wild Type = 136 bp

>chr12:104344449+104344584 136bp AGGGCCTAGAGTTGCTCCA TGTATTTTGGGTGCCAATGA

Sequence

Sequence (deletion in lower case)

 

AGTATATCAGAGACTGAAACACAAATGTACCCTTTGGGCTCCTCTTAGCATTGAGGAAGAAAGAGTGTTGCAATGAAAATTAAATTAACCATCATCAAATAGGTTAAAATGTGAGAAAGGGTAAAGTTAAGAAGGGGAGTGTCCAAAGATTAGGAAAGCTGGAATCTGATGCAGTGTGCGATTTTAAATAGAAGGCTCATTAGAAGATGTTTTAGGGTTGCCATGCTAGGGGAGAATTGTACTGTGGATGTAGGAGGAAAGCATACTAAGTCACAGAACGGCTAGGAACAAGTCAGGTTGCCTGTCCACTGTaaacactctcgggacctgttggatgattaatgcattagaaagagcaaagtcaagtcctctaggcatggataggaagaggattttctccagggagaaggctgtgtggtgaggagacccagcgacatgatggattgagggggggtcatagagtccagggactcttaccttgctcttttttattctcagccaaatagaggagctaaacctgcccaagttctccatcgctagtgactacaggctggaagaggatgtccttccagaaatggggattaaggaagtcttcacagaacaagctgacctatctgggatcacagaagccaagaaactgagtgtgtctcaggtaatacaagaagcacaggatgttttgccttgggtcctgaatgggggtggtgaagagcatgtaggctgtggggatggaacatgtgtgagaactacattgctgaggttccttaacaaatgtccatcttctacccctgtacataagctcacctaggagcccatagtggagagggcctagagttgctccatagaaccatggatactaacatgtgctgccctgcagggcaacatacagtacactgcaggggcacctacaatacactaCAGGGGCACCCACCATTCACTTCATTGGCACCCAAAATACATTGTAGGCCAAACCACTTCCATACTTTACCTCCTATACTACAGTGCTGGGACAGTACTTCTTGACCAGCACTCAGATTATTAATAGTAAAAGTAATGACAAGGAGTCATGGTGGTTTTGAGGAAAACTCCAAGAATGGGAAAGAAGATGCTTTCGACCCCTATACTACATTCTTAGGTGCACAGCCTGGCTGTAACCAATGTTCAGTCCAGCGCCCACTCAGAGCTCTGGGTACAGATCAGTCCCAGCAGGACATGGTATTTTGAGAATGTGAAACCATGGGTTGCA

603 bp deletion beginning at Chromosome 12 positive strand position 104,343,941 bp and ending after 104,344,543 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37781 TGT GGA TGT AGG AGG AAA GCA Mutant Forward A
37782 TGT ATT TTG GGT GCC AAT GA Common A
37783 AGG GCC TAG AGT TGC TCC A Wild type Forward A
37784 Fluorophore-1 CGG CTA GGA ACA AGT CAG GT Quencher-1 MUT Probe
37786 Fluorophore-2 CTA ACA TGT GCT GCC CTG C Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37781 0.40 uM
37782 0.40 uM
37783 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.