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Notes
Taqman qPCR protocols are run on an ABI 7500, 7700, 7900 or the Roche LightCycler
480. Use an appropriate instrument-specific Fluorophore/Quencher combination.
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= 129 bp
IPC = 124 bp
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| 74015 | CAA AGA GCA AGT GAC AAA TGT | Transgene Forward | A | |||
| 74016 | TTG CCC AAC TGG TCC TTT T | Transgene Reverse | A | |||
| 74089 | Fluorophore-1 | CAG TAG CCC AGA AGA CAG TGG AGG | Quencher-1 | MUT Probe | ||
| 74090 | CTA TTA CAA CAA CAG CAA CAC CT | Internal Positive Control Forward | A | |||
| 74091 | TGA GGG AAC CAC GGT ATT GA | Internal Positive Control Reverse | A | |||
| 74092 | Fluorophore-2 | CAG CTC TGC TAA CCC CAG CT | Quencher-2 | IC Probe |
Reaction A
| Component | Final Concentration |
|---|---|
| Kapa Probe Fast QPCR | 1.00 X |
| ddH2O | |
| 74015 | 0.40 uM |
| 74016 | 0.40 uM |
| 74090 | 0.40 uM |
| 74091 | 0.40 uM |
| Wt Probe | 0.15 uM |
| Mutant Probe | 0.15 uM |
| DNA |
Cycling
| Step | Temp °C | Time | Note |
|---|---|---|---|
| 1 | 95.0 | -- | |
| 2 | 95.0 | -- | |
| 3 | 60.0 | -- | |
| 4 | -- | repeat steps 2-3 for 40 cycles | |
| 5 | 40.0 | -- | Forever |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
Strains Using This Protocol
| Stock Number | Strain Name |
|---|---|
| 024338 | B6.Cg-Sncatm1Rosl Tg(SNCA*A30P)192Rwm/J |
| 023837 | B6.Cg-Tg(SNCA)OVX37Rwm Sncatm1Rosl/J |
| 038895 | B6;D2-Tg(Mbp-SNCA)1Ema/RoriJ |
| 038896 | B6;D2-Tg(Mbp-SNCA)29Ema/RoriJ |
| 029124 | C57BL/6-Gba1tm1.1Mjff Tg(Thy1-SNCA)15Mjff/J |
| 038021 | C57BL/6-Tg(Th-SNCA*)MI2Mgsp Del(6Snca)1Slab/J |
| 032799 | C57BL/6-Tg(Thy1-SNCA*E35K*E46K*E61K)3798Nuber/J |
| 018442 | C57BL/6N-Tg(SNCA)129Mjff/J |
| 017682 | C57BL/6N-Tg(Thy1-SNCA)15Mjff/J |
| 028808 | FVB(B6)-Tg(SNCA*)BXwy/J |
| 010710 | FVB;129S6-Sncatm1Nbm Tg(SNCA)1Nbm/J |
| 010788 | FVB;129S6-Sncatm1Nbm Tg(SNCA*A30P)1Nbm Tg(SNCA*A30P)2Nbm/J |
| 010799 | FVB;129S6-Tg(SNCA*A53T)1Nbm Sncatm1Nbm Tg(SNCA*A53T)2Nbm/J |
| 027227 | STOCK Gba1tm1Rlp Tg(SNCA*A53T)1Nbm Sncatm1Nbm Tg(SNCA*A53T)2Nbm/Mmjax |
| 027344 | STOCK Gba1tm1Rlp Sncatm1Nbm Tg(SNCA)1Nbm/Mmjax |
| 037870 | STOCK Sncatm1Rosl Tg(Cnp-SNCA)M2Vle/KlukJ |
| 16 strains use this protocol | |