Protocol 37672: Probe Assay - Rbm48<em1(IMPC)J> Alternate 2
Version 3.0

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

 


>chr5:3595585-3595734 150bp ACAATAGCGCTTCTCACAGG TCTGCCTAAAGACTACCACACTCC

 

Mut = 116 bp

Wt = 150 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case):

 ATACCGGGAGGGCCGACGCCCTCGCGCGGTCAAGGTAGAGTGACTCGGATTCTTCTCTCCCCTCGGCAGGGACACTAAATTCCTGTGAGCACTAGCTCCCCAAGGCCTGCCTCCATAAAGGCATGGCAGGGATGTTGTGAGATCCATCTCTAAGTGGAGGGGGCACGAAGAACCCGGGCCCCGGTACCAAAGGTTATCACTACCCTCGGTTAGCTGCTTACCAAACCTCCAACCCACTATTTTTAGCTTTGTATCCTATGTGTTTACAATAGCGCTTCTCACAGGGCaagcccagggtgagtgagtgttcagtggctgtttacattaacctctctggagtcagcttttggaaaagacgtctgttaagtggtttagtcagatacccttatgtggagtgtggtagtctttaggcagaagagtgacagtttagtgtgtatgttttgcagaccggattgtttcttgttttcaaagctccatttaaaagggggtgggaggaccgatttgccataggcttgtttgaagagtacatttaacgaggtctcctttgactttaggtatatacaatcaatttggagtctcagtacttactaatacaaggagttcctgcagtgggagccatgaaggaattagttgagcgttttgctctgtatggtgcgattgaacagtataacgctctagatgaatacccagcggaagattttacagaagtttatctcattaaatttgtgaaattacagagtgcaaggtatgtacaagttaagcagaactcctgttaaaatcttattcctgttgctgtcattttggccccaactgtttcccaaatgTACCCAAAGCACAGAAGCACTTCTTGCTCTCAGTCTTCCTGATTGGGGGGAGGAGGGGAGGGGACAGGGTTTATTTGGATAGCCTTAGCTGTGCTAGAATTAGTTCTGTAGACCAGGCTGGCCTTGAACTCAAAGATCCCTTTGCCTTTGCCTCTGACTCTCGAGTGCTGGGTTTAAAGGTATGCACCACTCCACCTCGCCTTCCAGTTTGTTTTTCACCAGGCCTCACATCTGCGACTTAATCACTCTAACAGTGA

This mutation is a 427 bp deletion beginning at Chromosome 5 position 3,595,181 bp and ending after 3,595,707 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49315 ACA ATA GCG CTT CTC ACA GG Common A
49316 TCT GCC TAA AGA CTA CCA CAC TCC Wild type Reverse A
51231 Fluorophore-1 CTT CCT GAT TGG GGG GAG G Quencher-1 MUT Probe
51232 Fluorophore-2 AGG GTG AGT GAG TGT TCA GTG G Quencher-2 WT Probe
51931 GGC TAT CCA AAT AAA CCC TGT Mutant Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
49315 0.50 uM
49316 0.50 uM
51931 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.