DNA ISOLATION PROTOCOL FROM BLOOD

(adapted by Wanda Iriarte)

Step 1 (for fresh blood)
Centrifuge 5 mL of whole blood at 2,500 rpm for 5 minutes.
ipette o! plasma and transfer the interphase "ontaining the
white "ells (bu!y "oat) to a "lean #.5 or 2 mL $ppendorf%
tube.
&dd #.0 mL of 'ed Cell Lysis (u!er (see , in)ert se)eral times,
"entrifuge at *,000 rpm for 2 min.
Step 1 (for hemolyed blood)
+ransfer 5 mL of whole blood to a "lean ,al"on% tube.
&dd 5.0 mL of 'ed Cell Lysis (u!er (#-#) and in)ert se)eral
times (for #0 se" apro.imatedly.
Centrifuge at /,000 rpm for #0 min.
Step !
0e"ant the supernatant and repeat the red "ell lysis for 2
more times until pelleted white "ells are "lear of hemoglobin.
Step "
&dd 100 2L of 0igest (u!er and resuspend pellet thoroughly.
&dd 5 2L of roteinase 3 (20 mg4mL), )orte. and digest at 51
5C o)ernight, mi.ing o""asionally.
Step #
&dd 5 2L of '6&se & (#0 mg4mL) and in"ubate at 78 5C for 70
min.
Cool sample to room temperature.
Step $
&dd 700 2L of otasium &"etate solution (7 9) to the digested
sample and in)ert se)eral times to mi. well.
In"ubate at : 20 5C for #0 min.
Centrifuge at #/,000 rpm for #0 minutes.
Step %
+ransfer supernatant to a new tube.
&dd 500 2L of isopropanol and mi. the tube ba"; and forth to
pre"ipitate the 06&.
In"ubate at : 20 5C for #0 min (to ma.imi<e 06&
pre"ipitation).
Centrifuge at #/,000 rpm for #5 min.
Step &
our o! supernatant and add 100 2l of ethanol (80 =) to
wash pellet.
Centrifuge for #0 min at high speed.
Step '
Carefully pour o! the ethanol and air dry the pellet in the
hood during 5 to #0 min.
'esuspend the 06& pellet in 200:700 2L of +ris (#0 m9, p>-
*.0)
>eat the samples at 15 5C for about # h.
9i. well to resuspend the 06&.
Red Cell Lys(s B)*er (Opt(o+ A)
To prep,re $-- mL of Red Cell Lys(s B)*er (N.
#
Cl/ -01#
M1 Tr(s B,se/ -0-1& M)/
7.8/5 g of 6>
/
Cl
#.02? g of +ris (ase
Red Cell Lys(s B)*er (Opt(o+ B)
To prep,re 1-- mL of Red Cell Lys(s B)*er (Tr(s B,se/ 1-
mM1 2Cl/ 1- mM1 M3Cl
!
/ 1- mM)/
0.#2# g of +ris (ase
0.085 g of 3Cl
0.207 g of 9gCl
2
@1>
2
A
D(3est B)*er

To prep,re $- mL of D(3est B)*er (Tr(s B,se/ 1- mM1
N,Cl/ -0# M1 SDS/ !41 5DTA/ -0-$ M)/
500 uL of +ris (# 9, p>- *)
/ mL of 6aCl (5 9)
5 mL of B0B (20 =)
5 mL of $0+& (0.5 9)
Refere+6es
C&islamiento y puriD"a"iEn del 06& de un plFsmido
re"ombinanteG (0epartamento de (ioHuImi"a y (iologIa
9ole"ular, Campus Jni)ersitario de 'abanales) : &urora
Kal)Fn CeLudo, 9anuel +eLada, &ntonio Camargo, MosN Ma)ier
>iguera, Oi"ente 9aris"al y $milio ,ernFnde< 'eyes, 2001.
C06& e.tra"tion proto"ol from blood JC using uregene:li;e
reagentsG (JC 0a)is Lab) : Muan 9edrano (2008).
CIsolation of high:mole"ular:weight 06& from mammalian
"ellsG (9ole"ular Cloning- & laboratory manual) : Bambroo;,
,rits"h and 9aniatis (#?*?).