Protocol 47174: Probe Assay - Csdc2<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

Mutant=  99 bp

Wild Type = 110bp

>chr15:81946469-81946578 110bp ACACAAGTTGAAAGGACAGT CATGCTAAACTGTGTGATTAGT

Sequence

MUT Sequence (large deletion, junction in UPPER case):

agaagcagggctggctctttcaggacttcctgtggccttagcctagagccccagagccacagcagtgatcatcagggttctgtgataatggttatcagctgatccctgcctgaccccagctcactgctcccagcatcCCctgcctcggtatcctcaccagaaaaacaaactaatcacacagtttagcatgtaggaggctttagctttaatccccagcaacaaaacaaaggactgtcggaatgtttgtttgcggagctaatgccaccttaaaaccaggcgtgagcacactgtggtggcgcacacctttgatcccagggcttgaaagacagaggcaggggg

 

This mutation is a a 2,287 bp deletion of Chr15:81,946,521-81,948,807 (GRCm38/mm10)

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
70012 ACA CAA GTT GAA AGG ACA GT Wild type Forward A
70013 CAT GCT AAA CTG TGT GAT TAG T Common A
70014 TTA TCA GCT GAT CCC TGC CT Mutant Forward A
70015 Fluorophore-1 ACT CCC GAA GAC ACT CAC Quencher-1 WT Probe
70016 Fluorophore-2 CCA GCA TCC CCT GCC T Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
70012 0.40 uM
70013 0.40 uM
70014 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.