Protocol 35310: Probe Assay - Lyve1<tm1.1(EGFP/cre)Cys> 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

Mutant=  115 bp

Wild Type = 110 bp

>chr7:110852071+110852180 110bp GAACTCTGCAGTCTTTTTCTGC GAAGTTTAGATGCAAGAGAGTGG

Sequence

Wt Sequence: GAACTCTGCAGTCTTTTTCTGCAGGAACTGACAGTGGCTTGCTTCTTTGGTTCCCGGGGCATGAAACTTGCCTCGTGTGCACCTTCTCCACTCTCTTGCATCTAAACTTC

 

Mutant Sequence: GAACTCTGCAGTCTTTTTCTGCAGGAACTGACAGTGGcgGTGGCGGCCGTCTGTTAATTAAGGTGTGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATTTGGTACCGG

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
45741 GAA CTC TGC AGT CTT TTT CTG C Common A
45742 CCG GTA CCA AAT TCC GAA G Mutant Reverse A
45743 GAA GTT TAG ATG CAA GAG AGT GG Wild type Reverse A
45744 Fluorophore-1 CGG CCG TCT GTT AAT TAA GGT Quencher-1 MUT Probe
45745 Fluorophore-2 CTG ACA GTG GCT TGC TTC TTT Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
45741 0.40 uM
45742 0.40 uM
45743 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.