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Stock No: 013234
Protocol 21365: Standard PCR Assay - Tg(Cd4-cre)1Cwi-Chr 3-alt1
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
Mut Tm = 88.5
WT Tm = 80.5
Mutant = 336 bp
Heterozygote = 153 bp and 336 bp
Wild type = 153 bp
This assay can distinguish hemi from hom- integration site is known to be on mouse Chr 3.
http://www.sciencedirect.com/science/article/pii/S002217591630120X
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 32288 | GTT CTT TGT ATA TAT TGA ATG TTA GCC | Common | A | |||
| 32318 | TAT GCT CTA AGG ACA AGA ATT GAC A | Wild type Reverse | A | |||
| 32324 | CTT TGC AGA GGG CTA ACA GC | Mutant Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 32288 | 0.50 uM |
| 32318 | 0.50 uM |
| 32324 | 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.