Multiplex Genotyping PCR for the KPC Pancreatic Cancer Mouse Model
Shou Kitahara, Shounak Ranabhor, Thae Su Thu, Neha Ramesh, Chang-il Hwang
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Abstract
Genetically engineered mouse model (GEMM) is one of the most important pre-clinical models in cancer research. In pancreatic cancer research, Kras+/LSL-G12D; Trp53+/LSL-R172H; Pdx-1-Cre +/LSL-G12D; Trp53+/LSL-R172H; Pdx-1-Cre (KPC) mouse model has been widely used in the laboratory setting, since it faithfully recapitulates the progression of human pancreatic ductal adenocarcinoma. Polymerase Chain Reaction (PCR) is commonly used to genotype GEMMs. However, genotyping one gene at a time is inefficient and labor intensive. To simplify the genotyping process, we multiplexed three separate genotyping PCR protocols for a single PCR reaction. In addition, we provided the optimized PCR protocol for determining heterozygous or homozygous status of Trp53LSL-R172H LSL-R172Hallele. Overall, this protocol offers cost-efficient and accurate genotyping results for KPC mice.
Attachments
Steps
Crude genomic DNA isolation
Prepare Lysis Buffer (Recipe for 10 mL)
●0.0121 g Tris-HCl pH 8.0
●0.0373 g KCl
●12.5 µl 2M MgCl2
●45 µL NP-40
●45 µL Tween-20
●9.9 mL Ultrapure Distilled Water (DW)
Add 3 µl Proteinase K (20 mg/mL) to 1 mL of Lysis buffer prior to use (Store in -20°C)
Add 30 µl Lysis buffer with Proteinase K to cut tissue in 1.5 mL tube
Incubate at 56°C for 1.5 hours
Place on ice briefly and spin down briefly in a tabletop centrifuge
Incubate at 96°C for 10 min in ThermoMixer
Polymerase Chain Reaction for KPC
Prepare PCR master mix for KPC in 1.5mL centrifuge tubes
- 10 µL Platinum hot start PCR Mix
- 0.4 µL 10 mM primer each (7 primers)
- 6.2 µL DW
Aliquot 19 µL of master mix into each PCR tube
Add 1 µL DNA template to individual PCR tubes
Perform PCR using the following conditions:a.94°C 3 min
b.94°C 1 min/ 65°C 2 min/ 72°C 1 min (40 cycles)
c.72°C 3 min / 4°C ∞
Polymerase Chain Reaction for Trp53 Het/Homo
Prepare PCR master mix for Trp53 het/homo in 1.5mL centrifuge tubes1. 2 µL 10X NEB polymerase buffer
- 0.4 µL 10mM dNTP
- 0.4 µL 10mM primer (4 primers)
- 0.1 µL Taq polymerase
- 14.9 µL DW
Aliquot 19 µL of master mix into each PCR tube
Add 1 µL DNA template to individual PCR tubes
Perform PCR using the following conditions:a.94°C 3 min
b.94°C 30sec/ 60°C 30sec/ 72°C 30sec (40 cycles)
c.72°C 3 min / 4°C ∞
Gel Electrophoresis
Make 2% agarose gel in TAE buffer
Add 2 µL ethidium bromide (10 mg/ml stock concentration) to 100 ml melted agarose. Swirl to mix.
Pour into the casting mold with combs inserted to set up wells.
Remove combs and casting mold from agarose gel after the cast harden (~ 30 min)
Place the gel into electrophoresis apparatus
Add 1X TAE buffer to electrophoresis apparatus until the gel is submerged
Add 4 µL 6X loading dye to each 20 µL PCR product and load 10 µL of it into the wells
Load 3 µL of 100 bp DNA ladder mixed with loading dye to 1st well of each lane
Run gel at 120V until loading dye is visualized 75-80 % down the gel
Remove the gel from the electrophoresis apparatus and visualize it under UV light
Analyze images to determine the genotype for each mouse
