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This assay will NOT distinguish hemizygous from homozygous transgenic animals.
Transgene =~440 bp
Internal positive control = 324 bp
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
oIMR3798 | AGG TGG ACC TGA TCA TGG AG | Transgene Forward | ||||
oIMR7338 | CTA GGC CAC AGA ATT GAA AGA TCT | Internal Positive Control Forward | ||||
oIMR7339 | GTA GGT GGA AAT TCT AGC ATC ATC C | Internal Positive Control Reverse | ||||
oIMR8346 | ATA CCG GAG ATC ATG CAA GC | Transgene Reverse |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTP KAPA | 0.26 mM |
Primer 1 | 0.50 uM |
Primer 2 | 0.50 uM |
Primer 3 | 0.50 uM |
Primer 4 | 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 |
---|---|
006148 | B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J |
004682 | B6.Cg-Tg(CAG-cre/Esr1*)5Amc/J |
008538 | B6.Cg-Tg(Cspg4-cre/Esr1*)BAkik/J |
036499 | C57BL/6-Tg(Aire-cre/Esr1)1Mmat/J |
024701 | D2.Cg-Tg(Plp1-cre/ERT)3Pop/SjJ |
037551 | NOD.Cg-Tg(Pdx1-cre/Esr1*)1Mga/MirmJ |
032429 | STOCK Krastm4Tyj Trp53tm1Brn Tg(Pdx1-cre/Esr1*)#Dam/J |
7 strains use this protocol |