For in-depth product & services help, ask our
Technical Information Scientists
Stock No: 017708
Protocol 29484: Standard PCR Assay - Rag2<tm1.1Flv>
Version 2.2
Notes
This assay does not work well without the use of a Hotstart Taq polymerase.
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 = 643 bp
Heterozygote = 643 bp and 404 bp
Wild type = 404 bp
WT primers designed in the deleted region. Mut primers flank the deleted region.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 11908 | AGG TCC AAG CTG CTG CCA C | Mutant Forward | A | Rag2 del | ||
| 11909 | GTG TGG GAT ATA GCT GTT GG | Mutant Reverse | A | Rag2 del | ||
| 11910 | GAG ATG TCC CTG AAC CCA GA | Wild type Forward | A | |||
| 11911 | TTG AGT GAG GAT TGC ACT GG | Wild type Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 11908 | 0.50 uM |
| 11909 | 0.50 uM |
| 11910 | 0.50 uM |
| 11911 | 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
| Stock Number | Strain Name |
|---|---|
| 017712 | C;129S4-Rag2tm1.1Flv Csf1tm1(CSF1)Flv Csf2/Il3tm1.1(CSF2,IL3)Flv Thpotm1.1(TPO)Flv Il2rgtm1.1Flv Tg(SIRPA)1Flv/J |
| 017708 | C;129S4-Rag2tm1.1Flv Csf1tm1(CSF1)Flv Il2rgtm1.1Flv/J |
| 017707 | C;129S4-Rag2tm1.1Flv Il2rgtm1.1Flv Tg(SIRPA)1Flv/J |
| 014593 | C;129S4-Rag2tm1.1Flv Il2rgtm1.1Flv/J |
| 4 strains use this protocol | |