Protocol 30706: Probe Assay - Asf1b<em1(IMPC)J> Probe
Version 1.0

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

>chr8:86488607+86488735 129bp CCCTCACAAGCTGATGTCAC TCTCCTGTGGGCCTAACTTG

Mutant=  148 bp

Wild Type = 129 bp

Sequence

Wt Sequence: ccctcacaagctgatgtcacaggactcaagtcagccTCtactatatggttcctctcggctactgagaggcctaattttcccgggagggccagagtcaaggtcacttggccaagttaggcccacaggaga

 

Mutant Sequence: ccctcacaagctgatgtcacaggactcaagtcagccTCtgaaagcacactggttttggcagtcaagtttaagttcaaatccttaaacttggactggtgacaaaactactgtaatcccagctacttaggatgtagaagcaggaagacca

526 bp deletion spanning ENSMUSE00001257874 (exon 2) beginning at Chromosome 8 positive strand position 83,964,745 bp CTACTATATGGTTCCTCTCG, and ending after CTAGAACCTTCCTCTTTTAC at 83,965,270 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
35236 Fluorophore-1 TCG GCT ACT GAG AGG CCT AAT Quencher-1 WT Probe
35237 CCC TCA CAA GCT GAT GTC AC Common A
35238 TCT CCT GTG GGC CTA ACT TG Wild type Reverse A
35239 TGG TCT TCC TGC TTC TAC ATC C Mutant Reverse A
35240 Fluorophore-2 ACA CTG GTT TTG GCA GTC AAG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
35237 0.40 uM
35238 0.40 uM
35239 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.