Protocol 45524: Probe Assay - Rnf222<em1(IMPC)J> Alt1
Version 2.0

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

Mutant=  108 bp

Wild Type = 120 bp

>chr11:68892243+68892362 120bp TGCTTGTCATAGCTGTGGTG CTCTCCTGAGACTGGCATCA

Sequence

Large deletion:  Mutant sequence with junction in uppercase

tgcttgtcatagctgtggtgaggcatcaggcacttcaggtatccatcCcactccacacacttcctacccaacccgaGGaaaggacagagaaattgatgggatggaaga

 

This mutation is a 3534 bp deletion beginning at Chromosome 11 position 68,892,320 bp and ending after 68,895,853 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
64284 TGC TTG TCA TAG CTG TGG TG Common A
64285 CTC TCC TGA GAC TGG CAT CA Wild type Reverse A
64286 TCT TCC ATC CCA TCA ATT TC Mutant Reverse A
64288 Fluorophore-1 CCA ACC CGA GGA AAG GAC AG Quencher-1 MUT Probe
65800 Fluorophore-2 CAC TGC TGG GAT GTC CTG TT Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
64284 0.40 uM
64285 0.40 uM
64286 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.