Protocol 36028: Probe Assay - Ppig<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

>chr2:69736202+69736293 92bp GGGTTTAGATGTGTCCTGTGC ACCGTAAGTGAGGCTGTGCT

Mutant=  105 bp

Wild Type = 92 bp

Sequence

Wt Sequence (deletions in lower case):

CAATACAACAGAACATCATACAACCAGGAATATTTAATAAAAACCTAGTGACAAGTAGAAATACAAGTGGATAGAAATAACTCTAAATGTCAACTGCATGCACTCACTGTCTTCAGCAgtcaataggtatggcttttatattccgaacaagcttttgtaaaactcgtatgggaaaccctttgcactcatgaggttctgctatataagggtatcacatgatttcttattttcagtacactttttttgtttttatagaagctaactttgtttttctgtttttattttatctgatgaatcccaaaagaacaacaaaaccaactcctcatttagatgggtaaagattattttaatatcttacaacatactttattttgttgtattagagtaagaagcttttatttaataatttttacatgacattttaggcatcatgttgtttttgggcaagtgatctctggtcaggaagttgtgagagagattgaaaaccagaaaacagatgccgctagcaaaccatttgctgaagttcggatactcagttgcggagagctgattccaaagtctaagggtaagaacgggtgcacagttaagggaactcttacttggataagtagagattataaataagcatgaggttttgtgggtttagatgtgtcctgtgctgtcagttttttctaatttatatgtgcttctgtacgtatgtGTCTGGGTCAAGCACAGCCTCACTTACGGTTCTTCAGAAGGCATGGACCATATACTGTAAGTACAATATACAATAAGAAAATTATAGTTGTGAAGAACTTTGT

This is a 582 bp deletion beginning at Chromosome 2 position 69,735,682 bp and ending after 69,736,263 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
47620 CCT AGT GAC AAG TAG AAA TAC AAG TGG Mutant Forward A
47621 GGG TTT AGA TGT GTC CTG TGC Wild type Forward A
47622 ACC GTA AGT GAG GCT GTG CT Common A
47623 Fluorophore-1 TTT ATA TGT GCT TCT GTA CGT ATG TGT CT Quencher-1 WT Probe
47624 Fluorophore-2 CAC TCA CTG TCT TCA GCA GTC TG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
47620 0.40 uM
47621 0.40 uM
47622 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.