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Stock No: 025672
Protocol 20345: Standard PCR Assay - Generic rtTA-Alternate 1
Version 5.0
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
TG = ~300 bp
Hemizygotes = ~300 bp and 415 bp
IPC = 415 bp
Assay cannot distinguish hemi from hom
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 21238 | CTG TCC CTG TAT GCC TCT GG | Internal Positive Control Forward | A | |||
| 21239 | AGA TGG AGA AAG GAC TAG GCT ACA | Internal Positive Control Reverse | A | |||
| oIMR8747 | CAT GTC CAG ATC GAA ATC GTC | Transgene Reverse | A | rtTA | ||
| oIMR8932 | CTG TTC GGC CTT GAA TTG AT | Transgene Forward | A | rtTA |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 21238 | 0.50 uM |
| 21239 | 0.50 uM |
| oIMR8747 | 0.50 uM |
| oIMR8932 | 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 |
|---|---|
| 023917 | B6.Cg-Lin28btm1.1Gqda/J |
| 034533 | B6.FVB-Tg(EmuSR-tTa)83Bop/DwfJ |
| 012433 | B6;C3-Tg(ACTA1-rtTA,tetO-cre)102Monk/J |
| 024851 | B6N.129S6-Sod2tm1(rtTA*M2,tetO)Jsg/J |
| 034802 | FVB/N-Tg(MMTV-tTA)25754Kuw/Mmjax |
| 005734 | NOD/Lt-Tg(Ins2-rtTA)1Ach/AchJ |
| 006999 | STOCK Dbttm1Geh Tg(Cebpb-tTA)5Bjd Tg(tetO-DBT)A1Geh/J |
| 024854 | STOCK Tg(Camk2a-tTA)1Mmay Fgf14Tg(tetO-MAPT*P301L)4510Kha/J |
| 003272 | STOCK Tg(Cebpb-tTA)5Bjd/J |
| 003273 | STOCK Tg(CMV-rtTA)4Bjd/J |
| 025672 | STOCK Tg(tetO-Fgfr2b/Igh)1.3Jaw/J |
| 026194 | STOCK Tg(Wnt1-rtTA2S*M2)1Whsu/J |
| 12 strains use this protocol | |