Oct1 (Pou2f1) conditional mouse genotyping protocol.
Arvind Shakya, 17 August 2015
Genotyping
Genotyping is performed using the following primers:
The presence of WT (364 bp) and floxed (493 bp) alleles is determined by PCR using Oct1-3'-WT and
Oct1-5'-flox primers, while the presence of the null (669 bp) allele is determined using Oct1-3'-WT and
Oct1-5'-null primers.
Oct1 3’WT: 5’-ACAGATGCTCGCATTTTAGTTG
Oct1 5’flox: 5’-ACACACACAAACAGCAATTATC
Oct1 5’null: 5’-TTGCCCAAGTCTTGTCTATCTG.
PCR was performed using a Bio-rad Tetrad2 Thermocycler.
Master mix was prepared as follows:
Ingredient
Initial Conc.
Volume
Final Conc.
2X
7.5 L
1X
Oct1 3’ WT
10 M
0.3 L
0.2 M
Oct1 5’ flox
10 M
0.3 L
0.2 M
GoTaq
Green
PCR
Mastermix
(M7123,
Promega)
dH2O
Tail DNA
WT/Flox PCR program
94°C (2 min)
94°C (30 sec), 49°C (1 min), 72°C (30 sec) X 30 cycles
72°C (10 min)
5.9 L
1 L
4°C (hold)
Null PCR:
Ingredient
Initial Conc.
Volume
Final Conc.
2X
7.5 L
1X
Oct1 3’ WT
10 M
0.3 L
0.2 M
Oct1 5’ Null
10 M
0.3 L
0.2 M
GoTaq
Green
PCR
Mastermix
(M7123,
Promega)
5.9 L
dH2O
1 L
Tail DNA
Null PCR program
94°C (2 min)
94°C (20 sec), 55°C (30 sec), 72°C (20 sec) X 30 cycles
72°C (10 min)
4°C (hold)
2