Protocol 31221: Probe Assay - Prim1<em1(IMPC)J> Probe
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.

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

>chr10:128016101+128016258 158bp GGCGCAGTATATTCTCACAGAG AAAGGTGCGTTTCATCAACA

Mutant=  149 bp

Wild Type = 158 bp

Sequence

Wt Sequence: GGCGCAGTATATTCTCACAGAgtaaggaatcttttttttcttttgtattttgtttgtatcttttttgtgtgtttattttataagccagagtgagttcttgcccccaagttttgaaGGtaatgttttagagtgtattattgttgatgaaacgcaccttt

 

Mutant Sequence: aaccctgtctcgaaaaaccaaaaaaaaaaaaaaaacaaaaaaaaaacgaacatatgtcttcccattagttaaggttttagttaaggttacattatgccctAGtaatgttttagagtgtattattgttgatgaaacgcaccttt

278 bp deletion beginning at Chromosome 10 negative strand position 128,016,216 bp.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
21666 AAC CCT GTC TCG AAA AAC CA Mutant Forward A
36408 GGC GCA GTA TAT TCT CAC AGA G Wild type Forward A
36409 AAA GGT GCG TTT CAT CAA CA Common A
36411 Fluorophore-1 AGT GAG TTC TTG CCC CCA AG Quencher-1 WT Probe
38421 Fluorophore-2 AGG TTT TAG TTA AGG TTA CAT TAT GCC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
21666 0.40 uM
36408 0.40 uM
36409 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.