Stock No: 021233
Protocol 22073: End Point Analysis Assay - Raf1<tm2.1Ara>End Point
Version 3.0

Notes

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 = A/A

Heterozygote =G/A


Wild type = G/G

 

Sequence

ATA TATTT CTCCA TGAAG GCCTC ACGGT GAAAA TTGGA (G/A)ATTT TGGTT TGGCA ACAGT GAAGT CACGC TGGAG TGGTT CTCAG CAGGT TGAAC AGCCC ACTGG CTCTG TGCT C TAG AT GGTAT ATAAA GAGGT ACTTC CGGAG TGCCA CTTTT AACCT CTA AA ACCAA ACCGT TGTCA CTTCA GTGCG ACCTC ACCAA GAGTC GTCCA ACTTG TCGGG TGACC GAGAC ACGA G ATCTA CC

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
16425 AAG TGA AGG AGG CCA GAA GC Forward A
16426 AGC TTC CTC ACC ATC CAC AG Reverse A
16427 Fluorophore-1 GAA AAT TGG AGA TTT TGG TTT G Quencher-1 WT Probe
16428 Fluorophore-2 GAA AAT TGG AAA TTT TGG TTT G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
16425 0.40 uM
16426 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 4.0 -- 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.