Protocol 43834: Probe Assay - Slc37a4<em3Lutzy>
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

>chr9:44400086+44400175 90bp GCTCCGCACGATTTTGTTAG CCACCCAATCTCCATTACCA

Mut= 87 bp

Wt= 90 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence:

TGTCTGCTGCTTATCCACAATGAACCTGCTGATGTTGGACTCCGAAATCTGGACCCTGCCCCCTCCAAGGGCAAAAAGGgtaagcctctatcaagctgactcctagagcaccttcctttgcccctggctccgcacgattttgttaggactagcctttgccttgccctgtgcttgccactggatgtaggcgaaggaatggtaatggagattgggtgggagaagtaaagattctatcctgaaatcagccagcgcatcctgagccttctctgacagcacagatctctccattctgggtaaccccctaagtttaagcagtttgggtgccttcttccacctgcaccgtcctcatttcctttcatttgcatcgaggtaggggagagggaagcagggctttgagggggtgccccacatcaatcctg

Mutant Sequence:

CTGGCCCCCCTGTGGGAAGATCCTGAGGAAGGTGAGTGCATCTGATGAGCCTGAGGGGGAGGGCACCCAGTGGGCACTTGGGAGCACCTCTTCTTACACAACCCTTCCACCCCTAACCCATAGCAGGTTCCTTAGGAGTTCCCTGCCAGCTCCTGCTGCTGTTGGGATATTTTAGGAGCCTGAGatGTAGGCGAAGGAATGGTAATGGAGATTGGGTGGGAGAAGTAAAGATTCTATCCTGAAATCAGCCAGCGCATCCTGAGCCTTCTCTGACAGCACAGATCTCTCCATTCTGGGTAACCCCCTAAGTTTAAGCAGTTTGGGTGCCTTCTTCCACCTGCACCGTCCTCATTTCCTTTCATTTGCATCGAGGTAGGGGAGAGGGAAGCAGGGCTTTG

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
64037 CCA CCC AAT CTC CAT TAC CA Common A
64048 GCT CCG CAC GAT TTT GTT AG Wild type Forward A
64059 TAG GAG TTC CCT GCC AGC TC Mutant Reverse A
64060 Fluorophore-1 CTA GCC TTT GCC TTG CCC Quencher-1 WT Probe
64061 Fluorophore-2 AGC CTG AGA TGT AGG CGA AGG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
64037 0.40 uM
64048 0.40 uM
64059 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.