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Stock No: 031435
Protocol 31724: Standard PCR Assay - Tcf7l2<tm1.1(EGFP/cre)Mrc>Mahll
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
>chr19:55742322+55742587 266bp CTGCTGCTGGTGGGTGA CTCTCCCGGCCCTGG
Mutant = 150 bp
Heterozygote = 150 bp and 266 bp
Wild type = 266 bp
At validation, a nonspecific band near the mutant band size was observed in the WT control. We will be using the melt protocol for genotyping, which clearly distinguishes all genotypes. Some troubleshooting may be required if this nonspecific product is observed on a gel.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 37498 | AGG TGG CAT CGC CCT C | Mutant Reverse | A | GFP | ||
| 37499 | CTC TCC CGG CCC TGG | Wild type Reverse | A | |||
| 37500 | CTG CTG CTG GTG GGT GA | Common | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 37498 | 0.50 uM |
| 37499 | 0.50 uM |
| 37500 | 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.