Protocol 37849: Probe Assay - Dnaaf3<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

>chr7:4528094-4528212 119bp GGAAGTCCCAGCTTCAATCA GAAGGTCTCACTCCGCTCTG

Mut = 113 bp

Wt = 119 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

GTTTTGGTTTGCTTTCAGATAGTTTTATTGTATTCCAGGGGCTATGTCACCCTTTTCTGATCTCTGAAAAATCTTGcccatggtgaacagatgtacacactcaggcacacacacacacacacacacacacacacacacacacacacacacaccgaataaaaagtttaatttattaaaagtatattttaaagacaagacttgagactatatgtcagcttgctttgcttttgggaagtcccagcttcaatcagcagtgctgggtggtggttgcagttcctggattggtgcagtttgtcaagtctccctccctgtttcatccttccctcctccagagcggagtgagaccttcctggagctgtgggggaacgcgctgctgcgaccctcggtagctgccttcctgcgcgctcaggctagccacctggctaatctggtcctggagcctgatcgcctggaggagcaactaccctggcttagtcttcggctgctcaaggtgccacagctgcccacctttgttggagggaagtggaatttgcttgaaatttgggtggctgcaggaagaatggaaatgagcagttcagaggaattgaggggctggagcaggctggcgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtcctaattccacaattccacctctgtaactcccactactgtggcctagGGTTAGGGGAGACTTTGCTCTCTGTGCCTCATTTTTTCCGCCCCCATAATCACTACCACCTAATGGGATGGAAGGGGAGCCCTCAC

This mutation is a 617 bp deletion beginning at Chromosome 7 position 4,527,750 bp and ending after 4,528,366 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
52323 GGA AGT CCC AGC TTC AAT CA Wild type Forward A
52324 GAA GGT CTC ACT CCG CTC TG Wild type Reverse A
52325 GGG GCT ATG TCA CCC TTT TC Mutant Forward A
52326 CTT CCA TCC CAT TAG GTG GT Mutant Reverse A
52327 Fluorophore-1 CTG GGT GGT GGT TGC AGT T Quencher-1 WT Probe
52328 Fluorophore-2 TTG GGT TAG GGG AGA CTT TGC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
52323 0.40 uM
52324 0.40 uM
52325 0.40 uM
52326 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.