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This assay will NOT distinguish hemizygous from homozygous transgenic animals.
Genotyping by PCR is performed to determine the absence or presence of the transgene. This strain has some variability in repeat size, therefore Southern blotting is performed to accurately determine the size, in kilobases, of the repeat within the transgene.
Transgene = 213 bp
Internal positive control = 415 bp
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
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 | |||
35766 | TCG AAA TGC AGA GAG TGG TG | Transgene Forward | A | |||
35767 | CTT CCT TTC CGG ATT ATA TGT G | Transgene Reverse | A |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTP KAPA | 0.26 mM |
21238 | 0.50 uM |
21239 | 0.50 uM |
35766 | 0.50 uM |
35767 | 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 |