DNA EXTRACTION: QIAGEN DNEASY TISSUE AND BLOOD KIT

(30-step)
Date/Time:
Sample labelled:

Total sample:

Checking materials

2
4

3
20ul Proteinase K
100ul blood
Into microcentrifuge tube
(not provided)

100ul PBS (50mM

potassium phosphate,
150mM NaCl, pH
7.2)

DO NOT THROW AWAY

REMAINING BLOOD

Mixed and
incubated
sample

200ul buffer AL (without

added methanol)
and
5 Mix using vortex,
incubate at 56OC for 10
minutes

7
Mix thoroughly by vortexing- to
yield homogenous solution

200ul ethanol (96-100%)

11
Discard flow through and
collection tube.

8
Pipette out
Mixed sample

9
Dneasy mini spin column
place in a 2ml collection
tube (provided)
10
Centrifuge for 1min at
8000rpm (>6000g)

15
Discard flow through and
collection tube.
12
Place Dneasy mini spin
column in a new 2ml
collection tube (provided)

13
500ul Buffer AW1

14
Centrifuge for 1min at
8000rpm (>6000g)

19
Discard flow through and
collection tube.
16
Place Dneasy mini spin
column in a new 2ml
collection tube (provided)

20
Place Dneasy mini spin
column in a cleaned
microcentrifuge tube (not
provided)

17
500ul Buffer AW2

18
Centrifuge for 1min at
14,000rpm (20,000g)
-to dry the DNeasy
membrane

22
Incubate at room
temperature for 1 min

21
200ul Buffer AE
NOTE:
Elution with 100ul
instead of 200ul)
increases the final DNA
concentration in the
eluiate but also deceases
the overall DNA yield

23
Centrifuge for 1min at
8000rpm (>6000g)
-to elute

24
Keep the flow through
(genomic DNA) 1st step
microcentrifugation
(back up)
WHILST

25
Place Dneasy mini spin
column in a cleaned
microcentrifuge tube (not
provided)

26
200ul Buffer AE

27
Incubate at room
temperature for 1 min

END
29
Keep the flow through
(genomic DNA) 2nd
step microcentrifugation
(analysis)

30
Discard Dneasy mini spin
column

If two genomic DNA have adequate DNA from NanoDrop

quantification, both can be used to analyse gene (one genomic
sample 2 genes; meaning twice freeze-thaw)
KIV to extract the same sample (whole blood) as back up

28
Centrifuge for 1min at
8000rpm (>6000g)
-to elute