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This assay will NOT distinguish hemizygous from homozygous transgenic animals.
Melting curve analysis is done using a Roche Light Cycler 480.
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 |
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 |
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 |
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 |