Protocol-at-a-glance (Rev. 04)
Plasmid DNA Purification (NucleoBond
®
Xtra Midi / Maxi)
Midi
Maxi
1
Cultivate and harvest
bacterial cells
4,500-6,000 x
g
15 min at 4°C
2
Cell lysis
high-copy / low-copy
high-copy / low-copy
Buffer RES
Buffer LYS
(Important: check Buffer LYS
for precipitated SDS)
8 ml / 16 ml
8 ml / 16 ml
12 ml / 24 ml
12 ml / 24 ml
3
Equilibration of the
column together with
inserted column filter
Buffer EQU
12 ml
Buffer EQU
25 ml
4
Neutralization
Buffer NEU
8 ml / 16 ml
Buffer NEU
12 ml / 24 ml
5
Clarification and
loading of the lysate
invert the tube 3 times
load lysate on NucleoBond
®
Xtra column filter
lysate is simultaneously cleared and loaded
onto the NucleoBond
®
Xtra column
6
1
st
Washing
Buffer EQU
5 ml
Buffer EQU
15 ml
7
Discard NucleoBond
®
Xtra column filter
discard NucleoBond
®
Xtra
column filter
discard NucleoBond
®
Xtra
column filter
8
2
nd
Washing
Buffer WASH
8 ml
Buffer WASH
25 ml
9
Elution
Buffer ELU
5 ml
Buffer ELU
15 ml
10
Precipitation
NucleoBond
®
Xtra
Midi
NucleoBond
®
Xtra
Midi Plus
NucleoBond
®
Xtra
Maxi
NucleoBond
®
Xtra
Maxi Plus
Isopropanol
3.5 ml
15,000 x
g
30 min at 4°C
Isopropanol
3.5 ml
load NucleoBond
®
Finalizer
Isopropanol
10.5 ml
15,000 x
g
30 min at 4°C
Isopropanol
10.5 ml
load NucleoBond
®
Finalizer Large
11
Wash and dry DNA
pellet
70% ethanol
2 ml
15,000 x
g
5 min at RT
5-10 min
70% ethanol
2 ml
/
3 x air
70% ethanol
5 ml
15,000 x
g
10 min at RT
10-15 min
70% ethanol
5 ml
/
6 x air
12
Reconstitute DNA
appropriate
volume of TE buffer
Buffer TRIS
200-800 µl
appropriate
volume of TE buffer
Buffer TRIS
400-1000 µl
MACHEREY-NAGEL GmbH & Co. KG
•
Neumann-Neander-Str. 6-8
•
D-52355 Düren
•
Germany
Tel.: +49 (0) 24 21 969 270
•
Fax: +49 (0) 24 21 969 279
•
e-mail: [email protected]