Protocol 37434: Probe Assay - Papola<em1(IMPC)J> Alternate 3
Version 3.0

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

>chr12:105805212+105805336 125bp TCTGCTGGTTTCTTGCATTG GCTCTGATACTTCTCACATCCAGT

Mutant=  110 bp

Wild Type = 125 bp

Sequence

Wt Sequence (deletions in lower case and insertions with carrots ^ctggttttgggagt^ (ctggttttgggagt insertion):

 CTAACATTTTTATAATTCCAGTTCTTAGATAACAAAAAAAAAAAAACCAGAAAGACCCTCTGCATAAATTAACAAATAACTGAGAACTTTGTTGTTCTGTCATTTGAATTGGTTTCCCTGC^ctggttttgggagt^tttactaacaaccattgtttgttaacagaatctcccacaatctgtaattgaaaatgttggagggaagatttttacatttggatcttacagactaggagtccacacgaaaggtaggtaatactttgcttattttgagctagtagaatttgcatttctttaagattttgagttattaatgactcataaacgagctttggttattgtggacgtgaatccttaagaagctgtctgtagctgccatctgtaatgcagtatagtaagtaatacgctcagttaaccagtggagtaaggtgtgttttctgtatctaagcactctgctggtttcttgcattggaaagtgtgggcacagagcaaattccactgtggtctatggACAATTCTCCATAGATTCCTTCTTTCTTTCTGAAGAGTATCACTGGATGTGAGAAGTATCAGAGCTCTAAGAAAGTATCGTCATTTTCTTGTCCTCATTTTTTTTATTGTAGATAGCTTATAAAATACATCTTAGCAAATACAGAATCAGTATGCCTGTAGCTACTGATCACATTGAATATTTACTTTAAATTTTT

This is a 373 bp deletion beginning at Chromosome 12 position 105,804,899 bp and ending after 105,805,271 bp (GRCm38/mm10). There is a 15 bp insertion (ACTCCCAAAACCAG) at the deletion site.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
47860 GTT GTT CTG TCA TTT GAA TTG G Mutant Forward A
47861 GCT CTG ATA CTT CTC ACA TCC AGT Common A
47862 TCT GCT GGT TTC TTG CAT TG Wild type Forward A
47864 Fluorophore-1 TGG GCA CAG AGC AAA TTC Quencher-1 WT Probe
51332 Fluorophore-2 CTG CCT GGT TTT GGG AGT AC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
47860 0.40 uM
47861 0.40 uM
47862 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.