Ron Lab


 

Detection of Alb-haA1 transgenic mouse allele by genomic DNA PCR analysis


DNA prep

Prepare genomic DNA using proteinase K method. (the NaOH method is less effective)


PCR primer
Sense haGADD34.1S 5' CGG GCT GCA GGA ATT CGA TCT CGT T 3'
Antisense haGADD34. 6AS 5' CTG TGT CCT CTC CAG GGC AAC AGA C 3'


PCR fragment

335 bp only in transgenic mouse


PCR solution
set ×1
ddH2O 18.05 µL
10× buf 2.5 µL
5 mM dNTP 1.25 µL
20 uM SP 0.5 µL
20 uM AP 0.5 µL
homemade Taq 0.2 µL
genomic DNA * 2 µL
  25 µL

homemade 10x buffer (500mM Tris pH 9.0, 200mM NH4SO4, 15mM MgCl2)

* DNA dilution (1/20-1/50) may increase sensitivity of PCR reaction


PCR protocol
Denature 96 ℃ 5 min

35 – 40
cycles
Denature 94 ℃ 1 min
Anneal 55 ℃ 1 min
Extend 72 ℃ 1 min
Extend 72 ℃ 7 min