Protocol 36543: Probe Assay - Naa35<em1(IMPC)J>Alternate1
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

>chr13:59595196+59595296 101bp TGCAAGCATAATTGAAAGCA TCCAGACTAAAAGGAAACATGC

 

Mut= 87 bp

Wt= 101 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case):

 CCGCTCGCCCCCGCCCATAGGATTTTTTGTTTGTTTGTTTCTCATTACCTACCGTTGTGAGCTACCATGTTGCTGGGAATTGAACTCATGACCTCTGGAAGAGCAGTCAGTGCACTTAACTGCTGAGCCATCTCTCCATCCCCAAAAAATGATTTTTAAATTTTTAATTCTTACTGGAATATTAAGATAACCATCTTAATTTGCTTGTTAGTTTTCAGTTCTGTGGTTTTATATTCTTTTTTCTTtctTTTTTTTTTTTTTTGATCCATTTCATAATGCAAGCATAATTGAAAGCATCactgtggtaattgagtatctagtcaatccttagaaaatgatttttaaattaatccaagcatgtttccttttagtctggaaacttaaactttattgcgctattaaagtaacttttctctctgattcttttagagctgaagttgggagaactgcttcatgataagctgtgagtatttgtggtgtagtagttgtggaaaactttaatctttttttttcaggtagaatattctttgaagtccaagtaaatttatgtttcaaatgagttgtacaatttctatttcagtttgggtgagctatcttgagtcacTGACATGGTTAAGATGTGAAACGCCAAACTGTTAGGATACTTTTCACGAAGATGGACATGCTGAACATGTGATTTTTGAAGACAGCTATTAGTTGTATGTCTAGTATGGTAGATTTATCAATGACTGATGCTGGTGTGGAATAAAATTGAATATCTCCAAGTGAACCAAAGGAAAGGAAATCCTGGCCTATATTTATACCTCGCTGTGCTGCTGCAGACACACTGTGGCCAGGC

This mutation is a 306 bp deletion beginning at Chromosome 13 position 59,595,218 bp and ending after 59,595,523 bp (GRCm38/mm10). In addition, there is a 3 bp intronic deletion (TCT) 41 bp before the larger deletion site. 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48068 TGC AAG CAT AAT TGA AAG CA Common A Common
48069 TCC AGA CTA AAA GGA AAC ATG C Wild type Reverse A Wt R
48071 Fluorophore-1 ACT GTG GTA ATT GAG TAT CTA GTC AAT CC Quencher-1 WT Probe
48072 Fluorophore-2 TGA AAC GCC AAA CTG TTA GGA Quencher-2 MUT Probe
49050 ATG TTC AGC ATG TCC ATC GT Mutant Reverse A Mut R

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48068 0.40 uM
48069 0.40 uM
49050 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.