Stock No: 026621
Protocol 35489: Probe Assay - Pus1<tm1.1Mdf> 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 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

>chr5:110777584-110777691 108bp CAGGCAGGTTGTATCCCTGA GTAGAAGCCGCAGGCAAGTA

Mutant=  146 bp

Wild Type = 108 bp

Sequence

Wt Sequence: CAGGCAGGTTGTATCCCTGAAAACCATGGCACAGATATGAGGAAGATGTCCTTCCAGCGGTGTGCTCGAACAGACaAGgtatgtgtcctacttgcctgcggcttctac

 

Mutant Sequence: GCCCAATTCCGATCATATTCAATAACCCTTAATATAACTTCGTATAATGTATGCTATACGAAGTTATTAGGTCCCTCGAGGGGATCCACTAGTTCTAGAGCGGCCGACAGCTGAaggtatgtgtcctacttgcctgcggcttctac

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
24716 GCC CAA TTC CGA TCA TAT TC Mutant Forward A
46207 CAG GCA GGT TGT ATC CCT GA Wild type Forward A
46208 GTA GAA GCC GCA GGC AAG TA Common A
46209 Fluorophore-1 AGG AAG ATG TCC TTC CAG C Quencher-1 WT Probe
46210 Fluorophore-2 TGT ATG CTA TAC GAA GTT ATT AGG TCC C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
24716 0.50 uM
46207 0.50 uM
46208 0.50 uM
Glycerol 6.50 %
Dye 1.00 X
Kapa 2G HS taq polym 0.03 U/ul
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.