Fission yeast genome preparation
Aim
prepare the genomic DNA of fission yeast for PCR template.
Materials
Zymolyase Buffer
1.2M Sorbitol
0.1M EDTA
14mM 2-ME
Zymolyase solution
15mg/ml Zymolyase-100T in Zymolyase Buffer
SDS-TE Buffer
0.5M EDTA 300ul
1M Tris-HCl (pH 7,5) 300ul
10% SDS 160ul
Methods
Suspend cells in 500ul Zymolyase Buffer. (1/8 plate cells are sufficient)
add 17ul 15mg/ml Zymolyase-100T.
incubate at 37℃ for 1-3h (In case of plasmid rescue, incubation time must not be extended.)
10krpm 2min at R.T.
remove sup and suspend in 0.3ml TE.
add 76ul SDS-TE buffer.
65℃ 30min
add 100ul 4M KCl on ice.
5min incubation on ice.
13krpm 5min at 4℃.
take sup and mix 1ml EtOH.
13krpm 20min at R.T
remove sup and wash with 100ul 70% EtOH
13krpm 5min at R.T
remove sup totally and dry up.
suspend pellete in 50ul TE.
For PCR template, 1ul DNA is used for 50ul scale PCR.
For plasmid rescue, 5ul DNA is used for the transformation with 50-100ul competent E.coli cell. (Competency of E.coli is important, at least 10^7 cfu/ug should be required. )
Remarks
y-goto.icon
Date :2017/08/03
Modified Date :
Author :y-goto.icon
Protocol template_2