Protocol 32280: Probe Assay - Cspp1<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

>chr1:10047381+10047512 132bp TTGTGGAGTTCTCCCCTTTG GGCTGTTGTTGGCTGCTT

Mut= 96 bp

Wt= 132 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 

CTACCTGGCTGATGAACTTCAAGATCTGTCTGTCTGTGATCTCTCAGAGTTTGGGTTACAGACACACGCTGCTGGGCCTGGCTTTTACATGGGTGTTGGTTTGGGAATACAAGCTCAGGTCCTCAGACTTGCACAGAGGGCACTTCACTGACTGAGCCGTCTCCTCATCCCCACTCTTTACATATCATGCATATGTAAATATTACATGCAAGTACAACATCACATAACTAAGAACTACATTTGTTTAGTGTctccagcctatcccctagcctcctggcccaatagcagaagcttcccaattttgtttttcactcttttctttctagtggctggcccagtgtacttaatagtaactcatttttaagaggatgaattaggtatcagaacagttgttacagtatgtaagtactttacacttcaatctctgtgctgtgtaattttttctaagttatttagtatgtcaaataagttaaattgtacattttgtggagttctcccctttgatttgtaaatatgtcttttccagggaaaggcgtcagagaaactctctgaaaacagtaaaatattaatctctatggctaaggagaacattccaccaagcagccaacaacagcccaagggacccttaggtatggtactgtatgcgtggaactaactttaagatatcctttctttgtagggcattgtgaaagaggtttgtttcttcagaaaaaagatttctttggatcttcatttttgagtcttacatatggcaatggggtttgtgtatgaaatgtaactcagagaacgttacctatttcattgatgttgcctaattgtgattggtggaaatgatattagaaaCTGTGTATGATGTAACTAAAAAATAGTAATAAAATTTTTAAGTGTGTTTATGTATATGTATACATGAGAGAGTCTGTATTGTTTTAAATTCAAAGAAGGGTCTAGCAAGATGGCTCAGCTGGTAAAGATGCTTGCCACCAAGCCTCAGGACCTGAGTTTGCCCCCCAGGACCCATATGGTGGTGTGCACACACATATACAAGTAAATGTGCTTA

 

This mutation is a 592 bp deletion beginning at Chromosome 1 positive strand position 10,047,149 bp and ending after 10,047,740 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
38734 TTG TGG AGT TCT CCC CTT TG Wild type Forward A
38735 GGC TGT TGT TGG CTG CTT Wild type Reverse A
38736 CCC CAC TCT TTA CAT ATC ATG C Mutant Forward A
38737 ACA TCA TAC ACA GAC ACT AAA CAA ATG Mutant Reverse A
38738 Fluorophore-1 TGG CTA AGG AGA ACA TTC C Quencher-1 WT Probe
38739 Fluorophore-2 ACA TGC AAG TAC AAC ATC ACA TAA CTA A Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
38734 0.40 uM
38735 0.40 uM
38736 0.40 uM
38737 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.