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Technical Information Scientists
Donating Investigator reports this transgene had integrated at locus 6qB3 of the mouse genome (nucleotide 56,524,796) and did not interrupt any known gene.
WT F and Common R primers amplify a PCR product from mouse of 303 bp. Tg F primer is specific for the Tg.
This assay will distinguish hemizygous from homozygous transgenic animals.
Changed common from .13791 (no amplification with Tg) to .13790 per Kai (successful amplification with Tg) SC 3-1-12.
Mutant = ~500 bp
Heterozygote = 303 bp and ~500 bp
Wild type = 303 bp
Melt Peaks
Wt = 82.7°C +/- 1
Tg = 82.6°C +/- 1
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
13790 | TGA AAT CCG GGT GGT ATT GG | Common | A, B | |||
13791 | GGT GAG TTT AAC CTT CAA GGG CT | Wild type | A | |||
13792 | AGC TTG CTA GCG GAT CCA GAC | Transgene | B |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTPS-kapa | 0.26 mM |
13790 | 0.50 uM |
13791 | 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 |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTPS-kapa | 0.26 mM |
13790 | 0.50 uM |
13792 | 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 |