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This assay will NOT distinguish hemizygous from homozygous transgenic animals.
Transgene = 180 bp
Internal positive control = 200 bp
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
oIMR5984 | GCT AAC CAT GTT CAT GCC TTC | Transgene Forward | A | CAG | ||
oIMR8744 | CAA ATG TTG CTT GTC TGG TG | Internal Positive Control Forward | A | |||
oIMR8745 | GTC AGT CGA GTG CAC AGT TT | Internal Positive Control Reverse | A | |||
oIMR9074 | AGG CAA ATT TTG GTG TAC GG | Transgene Reverse | A | Cre R |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTP KAPA | 0.26 mM |
oIMR5984 | 0.50 uM |
oIMR8744 | 0.50 uM |
oIMR8745 | 0.50 uM |
oIMR9074 | 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 |
---|---|
007966 | B6.Cg-Smn1tm3(SMN2/Smn1)Mrph/J |
004682 | B6.Cg-Tg(CAG-cre/Esr1*)5Amc/J |
019102 | B6N.Cg-Tg(CAG-cre/Esr1*)5Amc/CjDswJ |
017595 | FVB.Cg-Tg(CAG-cre/Esr1*)5Amc/J |
034859 | NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CAG-cre/Esr1*)5Amc/J |
034795 | NOD.Cg-Tg(CAG-cre/Esr1*)5Amc/J |
008783 | STOCK Grm7Tg(SMN2)89Ahmb Smn1tm3(SMN2/Smn1)Mrph Tg(CAG-cre/Esr1*)5Amc Tg(SMN2*delta7)4299Ahmb/J |
008813 | STOCK Trpa1tm2Kykw Tg(CAG-cre/Esr1*)5Amc/J |
004453 | STOCK Tg(CAG-cre/Esr1*)5Amc/J |
9 strains use this protocol |