Protocol 34742: Probe Assay - Pold4<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

>chr19:4232328+4232420 93bp TCCGATGAGCTGTCTGTCTG TGCCCTACCCTGGACTTAGA

Mutant=  106 bp

Wild Type = 93 bp

Sequence

TTGGCTAAAAAGGGAAGCTAGCATGTCTCGCACTTCCGATGAGCTGTCTGTCTGCTGGATGGCAAAGgcaaatgggtggggagattgccaggatatagagtcacagctctaagtccagggtagggcaaaggcctggatgtgcctgaggagccaccacatgttattgcaggggaagacACCCAgtccctcagccaggaggaaacagagctggagctgctgaggcagtttgacctggcctggcagtatgggccttgtacaggtgagcaccccatttccagtccctcccatcaggcacatctctgaatcagttctgttatcaatgtgtctcaaaagtaatcctgcctagctcttggacactgtctcctgaaactgactatggaggctcaagaaactgccgtccatgtccactatcctggattctcctgcctatcctgtcttcttttcctgatggcaggtatcacaaggctgcagcgctggagtcgggcagagcagatgggcttgaagccccccctagaggtgtaccaagtgttgaaggcacaccctgaagaccctcacttccaatgcaggtcaggataggctgggacagctttcaggggaaccagggacttctccaggagctgccaccctcTTGGTCTACGTAGGGGCTACCTTGACTTGGAGAAGTGGGAACAGACTTAGACGGTCCTTCCTGTCAAGCAAGAAGAGTCTGGCT

A 2-part deletion beginning at Chromosome 19 position 4,232,361 bp for 446 bp, followed by 5 bp of retained sequence (TGGGT), then an additional 110 bp deletion that ends after 4,232,921 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44356 TCC GAT GAG CTG TCT GTC TG Common A
44357 TGC CCT ACC CTG GAC TTA GA Wild type Reverse A
44358 CTT GAC AGG AAG GAC CGT CT Mutant Reverse A
44359 Fluorophore-1 AGA TTG CCA GGA TAT AGA GTC ACA G Quencher-1 WT Probe
44360 Fluorophore-2 AGA CCC ATT GGT CTA CGT AGG G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44356 0.40 uM
44357 0.40 uM
44358 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.