Protocol 43624: Probe Assay - Gng12<em1(IMPC)J>Alternate2
Version 3.0

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

>chr6:67015556+67015653 98bp GGTTTTGTAGATACTCTGGCACA TTGAAGGAGACTGGCTGCAT

Mutant=  97 bp

Wild Type = 98 bp

Sequence

Large deletion:  Mutant sequence with junction in uppercase

actgattcgtgctaaatttctgcagaaaggttttgtagatactctggcacatctagatcgtcaccaaaattaCGgatggcgtgaggaaggtggggtgctgacatttatcgctatccttgcagcat

This mutation is a 5857 bp deletion beginning at Chromosome 6 position 67,015,601 bp and ending after 67,021,457 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
62653 TTG AAG GAG ACT GGC TGC AT Wild type Reverse A
62654 ATG CTG CAA GGA TAG CGA TAA Mutant Reverse A
62655 Fluorophore-1 ATT TCT GGC TGG GAT ATT TGG Quencher-1 WT Probe
63347 GGT TTT GTA GAT ACT CTG GCA CA Common A
63519 Fluorophore-2 AGG AAG GTG GGG TGC TGA CA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
62653 0.40 uM
62654 0.40 uM
63347 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.