Protocol 36443: Probe Assay - Setbp1<em8(indel)Lutzy>
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=  86 bp

Wild Type = 80 bp 

>chr18:78857841-78857920 80bp TGTCCCCTGTGAGTGAGTCC GGAGGTGCTGTTGTTGTCTG

Sequence

Wt Sequence (with deletion in lowercase):
TTTGCGAGATTGGCTCACTGAAGGAGATAACGTTGTCCCCTGTGAGTGAGTCCCACAGCGAGGAGACTATCCCgagcgacagtggcatcgggacagacaacaacagcacctccgaccaagcagagaagagttcagaatcccggcggaggtactctttcgacttctgttcccTTGACAACCCAGAGGCCATTCCATCCGACACCAGCACAAAGAACCGGCACGGCCACCGGCAGAAGCATCTCATTGT

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
48580 TGT CCC CTG TGA GTG AGT CC Common A
48581 GGA GGT GCT GTT GTT GTC TG Wild type Reverse A
48585 Fluorophore-1 AGC GAC AGT GGC ATC GG Quencher-1 WT Probe
48778 CGG TTC TTT GTG CTG GTG TC Mutant Reverse A
48779 Fluorophore-2 AGA CTA TCC CTT GAC AAC CCA GA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
48580 0.40 uM
48581 0.40 uM
48778 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.