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Stock No: 003127
Protocol 18722: Standard PCR Assay - Generic Hygromycin Alternate1
Version 2.0
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
Tg = ~210 bp
IPC = 320 bp
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 21157 | CTC CCA ACC CCA GAG GTA GT | Internal Positive Control Forward | A | |||
| 21225 | AGA CCC CAG ATC CAG AAA GG | Internal Positive Control Reverse | A | |||
| oIMR1020 | CGG AAG TGC TTG ACA TTG G | Transgene | A | |||
| oIMR1021 | GTA TTG ACC GAT TCC TTG CG | Transgene | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| 21157 | 0.50 uM |
| 21225 | 0.50 uM |
| oIMR1020 | 0.50 uM |
| oIMR1021 | 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 |
|---|---|
| 030985 | B6.129P2(Cg)-Aph1atm1Bdes Aph1btm1Bdes Aph1ctm1Bdes/J |
| 029604 | B6.Cg-Col1a1tm1(CAG-Sirt2)Jmi/DsinJ |
| 035404 | B6;129S-Igs7tm166(tetO-EGFP*,CAG-tTA2)Hze/XwyJ |
| 034797 | B6;129S4-Gt(ROSA)26Sortm2(CAG-rtTA3,-mKate2)Slowe Col1a1tm1(tetO-GFP,-PRKAA1*)Rjsh/J |
| 034422 | B6;129S6-Slc17a7em1(flpo)Tasic/J |
| 014594 | C;129S4-Rag2tm1.1Flv Thpotm1.1(TPO)Flv Il2rgtm1.1Flv/J |
| 029931 | C57BL/6-Firretm1Rinn/J |
| 026872 | C57BL/6-Hamptm1Ddhf/J |
| 003127 | FVB;129P2-Bcl3tm1Ver/J |
| 035561 | STOCK Tc(HSA21,CAG-EGFP)1Yakaz/J |
| 10 strains use this protocol | |