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Stock No: 002709
Protocol 28551: Standard PCR Assay - Generic Luc
Version 5.2
Notes
This assay will NOT distinguish hemizygous from homozygous transgenic animals.
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
Transgene = 572 bp
Internal Positive Control = 200 bp
Internal Positive Control = 200 bp
Separated by gel electrophoresis on a 1.5% agarose gel.
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 9767 | GAA ATG TCC GTT CGG TTG GCA GAA GC | Transgene Forward | A | Luc | ||
| 9768 | CCA AAA CCG TGA TGG AAT GGA ACA ACA | Transgene Reverse | A | Luc | ||
| oIMR8744 | CAA ATG TTG CTT GTC TGG TG | Internal Positive Control Forward | A | |||
| oIMR8745 | GTC AGT CGA GTG CAC AGT TT | 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 |
| 9767 | 0.50 uM |
| 9768 | 0.50 uM |
| oIMR8744 | 0.50 uM |
| oIMR8745 | 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 |
|---|---|
| 006100 | B10.Cg-H2k Tg(NFkB/Fos-luc)26Rinc/J |
| 033605 | B6.Cg-S1pr1tm4.1Rlp Gt(ROSA)26Sortm2.1(luc*/Arrb2*)Rlp Tyrc-2J/J |
| 023429 | B6.Cg-Tyrc-2J Cdkn1atm1Hpw/J |
| 006098 | B6.Cg-Tg(Il2/NFAT-luc)83Rinc/J |
| 006101 | B6.Cg-Tg(TRE/Prl-luc)31FlvRinc/J |
| 002709 | B6;C3-Tg(TettTALuc)1Dgs/J |
| 027662 | C57BL/6-Tyrc-Brd Tg(Gh1-luc/EGFP)D8Mrln/J |
| 007800 | FVB/N-Tg(Ins1-luc)VUPwrs/J |
| 032442 | FVB/N-Tg(tetO/CMV-Dek,-luc)317Siwe/J |
| 012370 | FVB/NJ-Tg(Hspa1a-luc,-EGFP)2Chco/J |
| 10 strains use this protocol | |