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This assay will NOT distinguish hemizygous from homozygous transgenic animals.
Transgene = 215 bp
Internal positive control = 324 bp
IPC Tm = 82 Area = 33.5
Transgene Tm = 79.5 Area = 50 (WT* Tm = 83 Area = 28.5)
*MCA WT with Tg assay @TD50EG but expect minimal -to-no WT @TD60EG (TD60EG MCA plate was not exported). SC 6-28-12
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
14909 | AGA GGG AAG GCC TGA TGT GT | A | ||||
14910 | CAA GAT AGA CCA TAT CCT GCA CC | A | ||||
oIMR7338 | CTA GGC CAC AGA ATT GAA AGA TCT | A | ||||
oIMR7339 | GTA GGT GGA AAT TCT AGC ATC ATC C | A |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTP KAPA | 0.26 mM |
14909 | 0.50 uM |
14910 | 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 |
---|---|
023837 | B6.Cg-Tg(SNCA)OVX37Rwm Sncatm1Rosl/J |
018442 | C57BL/6N-Tg(SNCA)129Mjff/J |
010710 | FVB;129S6-Sncatm1Nbm Tg(SNCA)1Nbm/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 |
6 strains use this protocol |