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Stock No: 003890
Protocol 24870: Standard PCR Assay - Mecp2<tm1.1Bird>
Version 6.2
Notes
This assay does not work well without the use of a Hotstart Taq Polymerase.
The Mecp2 gene is X-LINKED. Males with the targeted allele are hemizygous mutants.
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
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 | A | |||
| 9877 | CCA CCT AGC CTG CCT GTA CT | Mutant Reverse | A | |||
| oIMR7172 | CTG TAT CCT TGG GTC AAG CTG | Wild type Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 9875 | 0.50 uM |
| 9877 | 0.50 uM |
| oIMR7172 | 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 | 94.0 | -- | |
| 2 | 94.0 | -- | |
| 3 | 65.0 | -- | -0.5 C per cycle decrease |
| 4 | 68.0 | -- | |
| 5 | -- | repeat steps 2-4 for 10 cycles (Touchdown) | |
| 6 | 94.0 | -- | |
| 7 | 60.0 | -- | |
| 8 | 72.0 | -- | |
| 9 | -- | repeat steps 6-8 for 28 cycles | |
| 10 | 72.0 | -- | |
| 11 | 10.0 | -- | hold |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.
Strains Using This Protocol
This is the only strain that uses this protocol.