Protocol 37384: Probe Assay - Gtf2h3<em1(IMPC)J>
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

>chr5:124583670+124583782 113bp TTCTTTGCCATGGGTTTAGG GGCAGTCTGGTGTCAAGTAGC

Mut = 119 bp

Wt = 113 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

CATGTGGCCCCACCATGTGTATATGATAGGTAAGGTTCTAATCTGTAAATGACTCTGTCTGTGAGGGGAAGAAGgtctcctctcagcttctttgccatgggtttaggaaaagaaaagctgagcagatttttagcagggaatccttgtcagatgtcattgagcatctgacaggactcgctgctacttgacaccagactgcctggggccacgtagcaagaccctttcttacacacaccacagtgtataataagtaaataatgctaagtaatagctactttttctttcaacgactcctccctcgaggctgatggggttgtgtgggagctgacatgttcctctcgtgtctgtcagtttactttatccaaatgcatggatgcagtgatggtgctggcgaattctcacctgttcatgaaccgctccaaccagctggctgtcatcgccagtcacattcaggaaaggtaggggccagaggctgcttttcagtcctttaacagttgcatgcatgggtagagcaccttccggttactcagaggatctttctcctccccctcagccggctcttatacccggggaagaacggtggacttggagacttcttcggagaccctggcaatgcgctccccgactgtaacccctctgggagtaaagatgggaaatacgagctgttgacagttgcaaacgaggtgatcgctgaggagatcaaggatctgatgaccaagagtaagagcctggctattgtcactgctgtctccatgtggtgggaattggtGTATGTTTTTAAAGTGCAAGATTCTTATGAATTCTTTAAGAATTTCATACATGTATGCCTTGTGTTTTGATGATATTCACCAATCCCATTCTTCTCCCTAACCCCAGATCCACTTTCATCTCCTCCCTACATCTCCTCTCCTCCCTACATCTCCT

This mutation is a 695 bp deletion beginning at Chromosome 5 position 124,583,657 bp and ending after 124,584,351 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
51208 TTC TTT GCC ATG GGT TTA GG Wild type Forward A
51209 GGC AGT CTG GTG TCA AGT AGC Wild type Reverse A
51210 CTC TGT CTG TGA GGG GAA GA Mutant Forward A
51211 GGA GAA GAA TGG GAT TGG TG Mutant Reverse A
51212 Fluorophore-1 CAG GGA ATC CTT GTC AGA TGT CA Quencher-1 WT Probe
51213 Fluorophore-2 CAT ACA TGT ATG CCT TGT GTT TTG ATG 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
51208 0.50 uM
51209 0.50 uM
51210 0.50 uM
51211 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.