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Stock No: 008215
Protocol 38211: Standard PCR Assay - Tg(TRAMP)8247Ng-Chr16
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
Mutant = 151 bp
Heterozygote = 151 bp and 264 bp
Wild type = 264 bp
>chr16:19272826-19273089 264bp CTGTACCAATGGGCTTGAGG GGCATTCAGCTTGGAGAAGA
This assay may be capable of distinguishing hemi from hom. Transgene insertion site is known to be on mouse Chr 16.
This assay is designed around this insertion site, but it has not been tested on hom animals.
This assay is NOT able to be used for copy number evaluation. If this is required, it is suggested to type by qPCR.
Sequence
5' integration site:
AATTGTGGAGTGGAAAGAGAGATTGGACAAAGAGTTTAGTTTGTCAGTGTATCAAAAAATGAAGTTTAATGTGGCTATGGGAATTGGAGTTTTAGATTGGCTAAGAAACAGTGATGATGATGATGAAGACAGCCAGGAAAATGCTGATAAAAATGAAGATGGTGGGGAGAAGAACATGGAAGACTCAGGGCATGAAACAGGCATTGATTCACAGTCCCAAGGCTCATTTCAGGCCCCTCAGTCCTCACAGTCTGTTCATGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATCCactagtTATGTTAAAAAAAAAAAGAATGGATCAATTTGCATTCTTCTCCAAGCTGAATGCCAGTTCAACCAGCACCATTTGTTGAGAACGCTGTCTTTTTTCCACGGATTGGTTTTAGCTCCTTTGACAAAGATCAAGTGACCATAGGTGTTTGGGTTCATTTATGGGTCTTGCATTCTATTCCATTTATCTAATTGTCTGTGATTGTACCAATACCATGGAATTTTTAATCACAATTTCTCAGTAATATAGCTTGAGGTCAGGAATGGTGATTTCCCCAAAAGTTCTTTTATTGTTGAGCATAGTTTTTGTTGTCCTGGCTTTTTTGTTATTCCAGGTGAATTTGCAAATTGATTTTTCAAACTCTATGAAGAATTGAGTTGGAATTTTGATGGGGATTGCATTGAATCTGTAGATTGCTTTCAGCAAGATGACAATTTTTACTATATTAACCCTGAAAATCCATGAGCTTAGGGAATCTTTCCATATTTTGAGATCTTCTTTCTT
Chromosome 16 sequence in blue
Junction is lowercase
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 53316 | CTG TAC CAA TGG GCT TGA GG | Wild type Forward | A | mChr16 | ||
| 53317 | GGC ATT CAG CTT GGA GAA GA | Common | A | mChr16 | ||
| 53318 | TCA CAA ATT TCA CAA ATA AAG CA | Mutant Forward | A | vector |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 53316 | 0.50 uM |
| 53317 | 0.50 uM |
| 53318 | 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.