PCR for Cre
Primers
Cre_F: GCCTGCATTACCGGTCGATGCAACGA
Cre_R: GTGGCAGATGGCGCGGCAACACCATT
Program
95ºC 5 min
35 cycles: 95ºC 30 sec - 63ºC 30 sec - 72ºC 30 sec
72ºC 5min
4ºC forever
Gel analysis
1.5-2% gel
TG band = 800 bp
Alternative protocol with an internal control:
Primers
CRE320F: GAACCTGATGGACATGTTCAGG
CRE320R: AGTGCGTTCGAACGCTAGAGCCTGT
MYOF: TTACGTCCATCGTGGACAGC
MYOR: TGGGCTGGGTGTTAGCCTTA
Program
95ºC 5 min
35 cycles: 95ºC 30 sec - 62ºC 30 sec - 72ºC 30 sec
72ºC 5min
4ºC forever
Gel analysis
2 % gel
Cre transgene = 320 bp
Myogenin internal control = 250 bp
PCR for Prox1-CreERT2
Primers
5Prox1_F: TGTCTGTGCCTCCATCTCAG
Prox1Cre_R: AGGCAAATTTTGGTGTACGG
Program
95ºC 5 min
30 cycles: 95ºC 30 sec - 60ºC 30 sec - 72ºC 30 sec
72ºC 5min
4ºC forever
Gel analysis
1.5-2% gel
TG band = 730 bp
PCR for Prox1-flox
Primers
Prox1-lx_F: TGCTGAAGATGTTGGTTGCT
Prox1-lx_R: GGCTTTTCTGTTGCTGAAGG
Program
95ºC 5 min
36 cycles: 95ºC 30 sec - 57ºC 45 sec - 72ºC 30 sec
72ºC 5min
4ºC forever
Gel analysis
2 % gel
WT allele about 100 bp
Floxed allele about 300 bp