Protocol 32849: Probe Assay - Mak16<em1(IMPC)J> Alternate1
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=  125 bp

Wild Type = 169 bp

>chr8:32276899-32277067 169bp CTGACTGGTCTGTGCAATCG GGTAGCACTGCCCTGAAGAG

Sequence

Wt Sequence (deletion in lower case):

 

AGTAGTTACTAACTATAAATGAACTTCTGTTGCTGAGTAATAGGGTATGGTCGTATCTGCATTTAGGTTTTTCTTGCGACTTTGTTGTTGTTTATAAGAATATATGTATTCATGTTAGGGGACGAGCGAGGCCTATTAAAAGATAGCTCTATCTTTTCATTCCCTAAAGACCCTTTCCTCACTGTGACACCCAGCAATGTGCTCTGACATGGTCCTCATCATCTTGTGTGTTTGTGCTTTGGTGGTGGTGTTGCTGgagcctacagtgagggcaaggattgagccatggagcaccagctgcaggccagagagcacggcctctttttatattaaacagcatctttggggccagagcaaaagggctttctttctgtcttgcaggtaatctggaatacactaggaaacaagcagttttgttctttcaaaataaggtgagtagattgcagcctagactaaaatgtgttcggccgcccgcttggttgtaaacatttctttccttgcatcctagaaccaagactcagggcttctgcagaaatgaatacagtctgactggtctgtgcaatcggtcgtcctgtcctctggcaaatagtcagtatgctaccattaaagaagagaaaggtacgggagctacacgacaatttataaacgGATGTCCCCAGCCTCTGTAAATAACAATAATAAAGCTGTCTAACTTCTCTTCAGGGCAGTGCTACCTGTACATGAAGGTTATAGAGCGAGCAGCTTTTCCCAGGCGTCTCTGGGAACGGGTAAGATTCAGTGAGAAAACTCTAGTGACCATTGGGCAAAAGCAAAATTGCCATGTAGGCCATCTCTGCACTTGAAAGCCACCCTCATAGATACCG

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
39027 CTG ACT GGT CTG TGC AAT CG Wild type Forward A
39028 GGT AGC ACT GCC CTG AAG AG Common A
39030 Fluorophore-1 TCC TGT CCT CTG GCA AAT AGT C Quencher-1 WT Probe
40126 TGT GCT CTG ACA TGG TCC TC Mutant Forward A
40127 Fluorophore-2 CTT TGG TGG TGG TGT TGC TG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
39027 0.40 uM
39028 0.40 uM
40126 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.