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Protocol received from:
Y. Eugene Yu, Ph.D.
Associate Professor
Department of Cancer Genetics
Center for Genetics and Pharmacology, Rm 2320
Roswell Park Cancer Institute
Elm and Carlton Streets
Buffalo, New York 14263
Targeted Mutation (duplication) = ~355 bp
Internal positive control = 324 bp
This is a targeted mutation (duplication) with no wild type-specific assay. Homs are lethal so only WT or het is possible.
Mutant Tm = 84.5 Area = 33 (WT Tm = 79.5 Area = 24 Tm = 84 Area = 12)
IPC Tm = 82 Area = 43
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
15357 | CTG CCA GCC ACT CTA GCT CT | Transgene Forward | A | m Chr16 | ||
15358 | AAT TTC TGT GGG GCA AAA TG | Transgene Reverse | A | HPRT | ||
oIMR7338 | CTA GGC CAC AGA ATT GAA AGA TCT | Internal Positive Control Forward | A | |||
oIMR7339 | GTA GGT GGA AAT TCT AGC ATC ATC C | 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 |
15357 | 0.50 uM |
15358 | 0.50 uM |
oIMR7338 | 0.50 uM |
oIMR7339 | 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 |