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Stock No: 010727
Protocol 23680: Standard PCR Assay - Bbs2<tm1Vcs>
Version 2.2
Notes
DI mut primers did not work on initial IMP Req55786 after multiple attempts w/various Taqs and conditions. J.Merriam asked 10/6/09 to email DI for specific protocol and mut control DNAs. KJ, MO, JM say 'ok' for generic neo on IMP samples while we wait for DI. SC 10/13/09.
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 = 300bp
Heterozygote = 300 bp and 593 bp
WT = 593bp
Heterozygote = 300 bp and 593 bp
WT = 593bp
Sequence
DI sent corrected primer sequence .9454 and known control DNAs.
(.9202 is not a valid primer). SC 11/09.
(.9202 is not a valid primer). SC 11/09.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 9199 | CAC CGA GGC TCA TCT ACA TTT | Wild type Forward | A | |||
| 9200 | TGC TTG CTG GTT TTC TTT TCT | Wild type Reverse | A | |||
| 9201 | GCT ACC CGT GAT ATT GCT GAA | A | ||||
| 9454 | GAG GCC AAT ATG TTT GGG TTA | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 9199 | 0.50 uM |
| 9200 | 0.50 uM |
| 9201 | 0.50 uM |
| 9454 | 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.