Protocol 37189: Probe Assay - Polr2g<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

>chr19:8797183-8797275 93bp AGGTGAAGTGGTGGATGCTG GAACCCTCACCTTAGGAAATACC

Mut = 117 bp

Wt = 93 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

CTCTGACTCGTTTGTGGTTTTTGAAAGACCTGTCTCAGCTTTGACCCTGGCAAGTCCTCCTGCCTTAGCCTACATGGTACTGAGAGAATAGGTGTATGTCAGCATATCTAGGCCTTCTTGGGCATTTACTGTACCATCATGAGAGCATCATTTAATATTTCATGTGCTAAGCTGGTCGGGGGTGGCGCATGCCTTTAATTCCAGCACTTgggaggcagaggcaggtggatttctgagttcaaggccagcctggtctaaagagtgagttccaggacagccagcgctacacagagaaactctgtctcgaaaaaacatatatatgtgtgtgtgtgtgtgtgtgtgtgtgtatatatatatatatatatttaggtgctaatctgtgtcctgtctcttcttgatcaatgaaccatctgaacagaggttctttcacacagagactcttattgctggtggcttttcagaggctcttcacatagctgtcttctttcatttttcatgcagatatggctttgtaattgctgtcaccaccatcgacaatattggtgctggtgtgatccagccaggccgaggttttgttctttatccagtgaaatacaaagctattgttttccggccctttaaaggtgaagtggtggatgctgtggtcactcaggtcaacaaggtaagagtattattcatagaaaggttatGGGGTATTTCCTAAGGTGAGGGTTCAGTCCCTAGTCCACTTACTAGTTGCCTTAAGTCTTTTGTTGTTGTTAAGATTCATTTTATCTTAGTTTTTTGGTTTTTCTTTTCTGGTTTTAGGGTTTTCTTTTTTCTTTTCTTTTCTTTTCTTTTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTGAGACAGGGTTTCTCTGTGTAGCCCTGGCTCTCCTGGAACTCACTCTGta

This mutation is a 480 bp deletion beginning at Chromosome 19 position 8,797,208 bp and ending after 8,797,687 bp (GRCm38/mm10). There is a single bp insertion A at the deletion site.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
50762 GAA CCC TCA CCT TAG GAA ATA CC Common A
50763 AGG TGA AGT GGT GGA TGC TG Wild type Forward A
50764 TGG GCA TTT ACT GTA CCA TCA Mutant Forward A
50765 Fluorophore-1 TCA CTC AGG TCA ACA AGG TAA GAG T Quencher-1 WT Probe
50766 Fluorophore-2 CAT GCC TTT AAT TCC AGC ACT TAG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
50762 0.50 uM
50763 0.50 uM
50764 0.50 uM
Glycerol 6.50 %
Dye 1.00 X
Kapa 2G HS taq polym 0.03 U/ul
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.