Protocol 43567: Probe Assay - Pacs1<em5/6Lutzy>
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

>chr19:5160196-5160313 118bp TGGGCAGATAACAGAGAAAATG TGGTTGTAAGAGTGATTGGTCAG

Mutant=  123 bp

Wild Type = 118 bp

Sequence

Wt Sequence:

ggtccacccttctcccttcatccctcttagaacatctctttcgtcataatcctggtgactgtgttgaatcacatagagaaaacatctgggcagataacagagaaaatgatgtttctaagcttatgtagacctaccagcaccctgcagtaccaggccctctggcatttctttctttctttctctgaccaatcactcttacaaccaagagacgctaaagccactaacactcagtcctctagc

Mutant Sequence:

aaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggatccccatcaagctgatccggaacccttaatataacttcgtatagcatacattatacgaagttatTCTGGCATTTCTTTCTTTCTTTCTCTGACCAATCACTCTTACAACCAAGAGACGCTAAAGCCACTAACACTCAGTCCTCTAGCTGGACCTGAACCACCAAGGTTCTGGTTGCT

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
25183 ATC ATG TCT GGA TCC CCA TC Mutant Forward A
51677 Fluorophore-1 AAG CTG ATC CGG AAC CCT TAA Quencher-1 MUT Probe
63392 TGG GCA GAT AAC AGA GAA AAT G Wild type Forward A
63393 TGG TTG TAA GAG TGA TTG GTC AG Common A
63394 Fluorophore-2 CCA GCA CCC TGC AGT ACC Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
25183 0.40 uM
63392 0.40 uM
63393 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.