Protocol 37500: Probe Assay - H2-K1<tm1Bpe> Probe Alternate5
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=  96 bp

Wild Type = 78 bp

Sequence

Wt (NOD background) Sequence: GACCGCGGGGTCCCGCAGGCTCTCACACGTTCCAGCGGATGTTCGGCTGTGACGTGGGGTCGGACTGGCGCCTCCTCCGCGGGTACCAGCAGTTCGCCTACGACGGCCGCGATTACATCGCCCTGAACGAAGACCTGAAAACGTGGACGGCGGCGGACACGGcggcgctgatcaccagacGCAAGTGGGAGcaggctggtgatgcagagtattacAGGGCCTACCTAGAGGGCGAGTGCGTGGAGTGGCTCCGCAGATACCTGGAGCTCGGGAATGAGACGCTGCTGCGCACAGGTGCAGGGGCCGCGGGCAGCTCCTCCCTCTGCCCTCGGGCTGGGGCTCAGTCCTGGGGAAGAAGAAACCCTCAGCTGGGTGATGCCCCTGTCTCAGAGGGGAGAGAGTGTCCCTGGGTCTCCTGATTCCTCATCACAGGGACTGCACTGAGTCTCCCAGGACTCAGCCCTCTCCCTGGACAGTGCCCAGGCTGTCTCAGGAGGGAAGGAGAGAATTTCCCTGAGGTAACAACAGCTGCTCCCTTCAGTTCCCCTGCAGCCTCTGTCAGCCATGGCCTCTCCCAGGCCGG

 

Mutant Sequence: ctttgctttccCtggttaagcagtacagccccaaaatggttaaggttgcaagCTTGCTGGTGAAAAGGACCTCTCGAAGTGTTGGATACAGGCCAGACTttgttggatttgaaattccagacaa

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49452 AGC AGT ACA GCC CCA AAA TG Mutant Forward A
49453 CTG GCC TGT ATC CAA CAC TTC Mutant Reverse A
49454 Fluorophore-1 AGG TTG CAA GCT TGC TGG TG Quencher-1 MUT Probe
50991 CTG AAC GAA GAC CTG AAA ACG Wild type Forward A
51486 CCT GTA ATA CTC TGC ATC ACC AG Wild type Forward A
51487 Fluorophore-2 CGG CGC TGA TCA CCA GAC Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
49452 0.40 uM
49453 0.40 uM
50991 0.40 uM
51486 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 4.0 -- Forever
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

Stock Number Strain Name
025216 NOD.Cg-Prkdcscid H2-K1tm1Bpe H2-Ab1em1Mvw H2-D1tm1Bpe Il2rgtm1Wjl/SzJ
023848 NOD.Cg-Prkdcscid H2-K1tm1Bpe H2-D1tm1Bpe Il2rgtm1Wjl/SzJ
2 strains use this protocol