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This assay will NOT distinguish hemizygous from homozygous transgenic animals.
Transgene = 288 bp
Internal positive control = 200 bp
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
9703 | AGG ACG GCG AGT TCA TCT AC | Transgene Forward | A | mCherry | ||
9704 | TGG TGT AGT CCT CGT TGT GG | Transgene Reverse | A | mCherry | ||
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 |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTP KAPA | 0.26 mM |
9703 | 0.50 uM |
9704 | 0.50 uM |
oIMR8744 | 0.50 uM |
oIMR8745 | 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 |
---|---|
029540 | B6.129P2(Cg)-Cd19tm1.1Oao/J |
028624 | B6.Cg-S1pr3tm1.1Hrose/J |
025525 | B6.Cg-Tg(Cx3cl1/mCherry)1Jung/J |
030748 | B6.Cg-Tg(Krt18-RFP)1Jsaw/J |
030745 | B6.Cg-Tg(Krt5-ECFP)1Jsaw/J |
031693 | B6.FVB-Tg(CAG-EGFP,-Sumo1,-Sumo2,-Sumo3,-mCherry)10Weiy/J |
029899 | B6;129S6-Gt(ROSA)26Sortm2(CAG-NuTRAP)Evdr/J |
025406 | B6;FVB-Tg(RP23-370F21-GCaMP3*/mCherry)1Mik/J |
024046 | C57BL/6-Tg(Csf1r-HBEGF/mCherry)1Mnz/J |
021423 | C57BL/6J-Tg(Dlx2-cre,-mCherry)9Grsr/GrsrJ |
021853 | FVB-Tg(Myh6-mCherry/Map1lc3a)1Rag/J |
018067 | FVB-Tg(Prism)1849Htz/J |
018071 | FVB-Tg(Prism)1861Htz/J |
029429 | STOCK Or8a1tm26Mom Tg(Or8a1,-MAPT/mCherry)5MGaFeins/J |
014177 | STOCK Tg(Afp-mCherry)1Hadj/J |
025693 | STOCK Tg(CAG-GFP,-mCherry/Pxn)#Mgin/KleyJ |
029600 | STOCK Tg(Or8a1-OR1A1,-MAPT/mCherry)D736Feins/J |
017470 | STOCK Tg(Tnc-mCherry)2Pmay/J |
011108 | STOCK Tg(Ttr-RFP)1Hadj/J |
19 strains use this protocol |