Stock No: 003890
Protocol 4197: Standard PCR Assay - Mecp2<tm1.1Bird>
Version 3.0

Notes

This assay does not work well without the use of a Hotstart.

The Mecp2 gene is X-LINKED. Males with the targeted allele are hemizygous mutants.
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 = 240 bp
Heterozygote = 240 bp and 465 bp
Wild type = 465 bp
Separated by gel electrophoresis on a 1.5% agarose gel.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
9875 AAA TTG GGT TAC ACC GCT GA Common
9877 CCA CCT AGC CTG CCT GTA CT Mutant Reverse
oIMR7172 CTG TAT CCT TGG GTC AAG CTG Wild type Reverse

Reaction A

Component Final Concentration
ddH2O
KAPA Rxn Buffer Contains 1.5 mM Mg2+ final concentration. 1.00 mM
dNTP 0.20
9875 1.00
9877 1.00
oIMR7172 1.00
DNA Loading Dye 0.69
KAPA LR HotStart Taq Initial Denature 3 Min This is a two enzyme system. One with proofreeading the other without. HotStart is provided by antibody. 0.01 mM
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 65.0 --
4 72.0 -- repeat steps 2-4 for 35 cycles
5 72.0 --
6 10.0 -- hold
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.