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This assay will NOT distinguish hemizygous from homozygous transgenic animals.
Conversion/LC Validation data attached to rec 161125
Transgene = 213 bp
Internal positive control = 415 bp
Transgene Tm = 83.5
IPC Tm = 89
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
13582 | CCA CCA CTT CCT GCC TAC AT | Transgene Forward | A | |||
13583 | CCA GAC CGT CTC CTT CTT TG | Transgene Reverse | A | |||
21238 | CTG TCC CTG TAT GCC TCT GG | Internal Positive Control Forward | A | |||
21239 | AGA TGG AGA AAG GAC TAG GCT ACA | 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 |
13582 | 0.50 uM |
13583 | 0.50 uM |
21238 | 0.50 uM |
21239 | 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 |
---|---|
030434 | NOD.Cg-H2-Ab1b-tm1Doi Tg(HLA-DRA,HLA-DRB1*0301)#Gjh/DvYtomJ |
021885 | NOD.Cg-Prkdcscid H2-Ab1b-tm1Doi Il2rgtm1Wjl/SzJ |
017637 | NOD.Cg-Prkdcscid Il2rgtm1Wjl H2-Ab1b-tm1Doi Tg(HLA-DRB1)31Dmz/SzJ |
029295 | NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(H2-Ea-HLA-DRB1*0401*)1Dv/SzJ |
4 strains use this protocol |