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Stock No: 028289
Protocol 25567: Standard PCR Assay - Dp(16Lipi-Zfp295)1Yey
Version 2.2
Notes
Protocol received from:
Y. Eugene Yu, Ph.D.
Associate Professor
Department of Cancer Genetics
Center for Genetics and Pharmacology, Rm 2320
Roswell Park Cancer Institute
Elm and Carlton Streets
Buffalo, New York 14263
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
Targeted Mutation (duplication) = ~355 bp
Internal positive control = 324 bp
This is a targeted mutation (duplication) with no wild type-specific assay. Homs are lethal so only WT or het is possible.
Mutant Tm = 84.5 Area = 33 (WT Tm = 79.5 Area = 24 Tm = 84 Area = 12)
IPC Tm = 82 Area = 43
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 15357 | CTG CCA GCC ACT CTA GCT CT | Transgene Forward | A | m Chr16 | ||
| 15358 | AAT TTC TGT GGG GCA AAA TG | Transgene Reverse | A | HPRT | ||
| oIMR7338 | CTA GGC CAC AGA ATT GAA AGA TCT | Internal Positive Control Forward | A | |||
| oIMR7339 | GTA GGT GGA AAT TCT AGC ATC ATC C | Internal Positive Control Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 15357 | 0.50 uM |
| 15358 | 0.50 uM |
| oIMR7338 | 0.50 uM |
| oIMR7339 | 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.