Protocol 34149: Probe Assay - Rasgef1b<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

>chr5:99231903-99231996 94bp CCTGAAAACTCTACTCTCTTTAATCC GGAAAGACTGTATGTACTTGGCACT

Mut= 83 bp

Wt= 94 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 GTGGACAGTTAAAAGTAACCAGCAGACCCTTCTTTGGAAGTGAGGGTCTGAGGGCACAGGCCTCCCAGGATAACATCATTTCAGAGAAACTTCAGTTCTTGGGAGGGAAGGAAGGACACGTTTGGGAGCGCATCCATgcgcattcacaggcttatctgtccacgttgcttccactgtagagttgctacagcgaacggaagaaaacacggaacttggaggcgtacgtagaatggtttaatcgtctaagctacttggttgctacagaaatctgcatggtgagacaacacattgttttttcctcagatccagttttcctgctttatcttctatctgttgggcctgtgtgctgggttcctgtctgctcacgctcgtttttcttacagcctgtgaagaagaagcaccgagcgaggatgatcgagtatttcatcgatgtggctcgagagtgtttcaacattggaaacttcaattctttgatggcaatcatctgtgagtattcgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtataaacactagttttatggggcttggaaattgtatccactgaaaatctgcctttcctgaaaactctactctctttaatccagagttattatgaaacgttcagaatgctttatGGACCTAAACTAGTGCCAAGTACATACAGTCTTTCCTTAAAGGACATTCATTGAATGTCTTTAGTTCATCTATGCTCAGTTAAATATCCATCTGTAAAAGCTGAGAACTGAAACACTTGTCTAAATACAAGGTTTCTAGAGGTAT

This mutation is a 501 bp deletion beginning at Chromosome 5 position 99,231,939 bp and ending after 99,232,439 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
42929 CCT GAA AAC TCT ACT CTC TTT AAT CC Wild type Forward A
42930 GGA AAG ACT GTA TGT ACT TGG CAC T Common A
42931 TTC AGT TCT TGG GAG GGA AG Mutant Forward A
42932 Fluorophore-1 AGA GTT ATT ATG AAA CGT TCA GAA TGC Quencher-1 WT Probe
42933 Fluorophore-2 AGC GCA TCC ATG GAC CTA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
42929 0.40 uM
42930 0.40 uM
42931 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.