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This assay will NOT distinguish hemizygous from homozygous transgenic animals.
Transgene = ~550 bp
Internal positive control = 324 bp
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
25348 | GAC CTG GAT GCT GAC GAA GG | Transgene Forward | A | Camk2a | ||
27058 | GCA GCT CTA ATG CGC TGT | Transgene Reverse | A | tTA | ||
oIMR7338 | CTA GGC CAC AGA ATT GAA AGA TCT | Internal Positive Control Forward | A | |||
oIMR7339 | GTA GGT GGA AAT TCT AGC ATC ATC C | Internal Positive Control Reverse | A |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTP KAPA | 0.26 mM |
25348 | 0.50 uM |
27058 | 0.50 uM |
oIMR7338 | 0.50 uM |
oIMR7339 | 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 |
---|---|
016198 | 129S6.Cg-Tg(Camk2a-tTA)1Mmay/JlwsJ |
024115 | B6.Cg-Igs7tm94.1(tetO-GCaMP6s)Hze Tg(Camk2a-tTA)1Mmay/J |
007004 | B6.Cg-Tg(Camk2a-tTA)1Mmay/DboJ |
003010 | B6;CBA-Tg(Camk2a-tTA)1Mmay/J |
025105 | FVB.Cg-Tg(Camk2a-tTA)1Mmay/DboJ |
024108 | STOCK Igs7tm93.1(tetO-GCaMP6f)Hze Tg(Camk2a-tTA)1Mmay/J |
024854 | STOCK Tg(Camk2a-tTA)1Mmay Fgf14Tg(tetO-MAPT*P301L)4510Kha/J |
7 strains use this protocol |