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Stock No: 018785
Protocol 28502: Standard PCR Assay - Tg(LRRK2)
Version 5.2
Notes
This assay will NOT distinguish hemizygous from homozygous transgenic animals.
Melting curve analysis is done using a Roche Light Cycler 480.
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 = 154 bp
Internal Positive Control = 324 bp
Internal Positive Control = 324 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 | |||
| 8752 | TGA TTC TCG TTG GCA CAC AT | Transgene Forward | A | LRRK2 | ||
| 8753 | GCC AAA GCA TCA GAT TCC TC | Transgene Reverse | A | LRRK2 |
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 |
| 8752 | 0.50 uM |
| 8753 | 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 |
|---|---|
| 013583 | B6.Cg-Tg(tetO-LRRK2)C7874Cai/J |
| 012445 | B6.FVB-Tg(LRRK2)WT1Mjfa/J |
| 012446 | B6.FVB-Tg(LRRK2*G2019S)1Mjfa/J |
| 021913 | B6.FVB-Tg(LRRK2*Y1699C)C7.11HMjfa/J |
| 013725 | B6;SJL-Tg(LRRK2)66Mjff/J |
| 018785 | C57BL/6J-Tg(LRRK2*G2019S)2AMjff/J |
| 018786 | C57BL/6J-Tg(LRRK2*R1441G)3IMjff/J |
| 009604 | FVB/N-Khdrbs2Tg(LRRK2*R1441G)135Cjli/J |
| 009610 | FVB/N-Tg(LRRK2)1Cjli/J |
| 009609 | FVB/N-Tg(LRRK2*G2019S)1Cjli/J |
| 10 strains use this protocol | |