Edge Lab Agilent Hybridization Protocol 5/5/11

Adapted from Agilent One-Color Microarray-Based Low Input Quick Amp Labeling Protocol
otes!
-"#en working wit# labeled $A% work &uickly and in t#e dark% as Cy-' is p#otoreacti(e)
-$eagent*+n,yme product information found at end of protocol)
--eat water bat# to ./C at least 0 #our before starting)
Step 1: Prep Blocking Agent 12 3 minutes4
10x Blocking Agent can be prepared in advance & stored @ -20 C for up to 2 mo. After ta!ing" vortex
& centrifuge for #-10 s. $nl% needs to be prepared !en opening fres Blocking Agent
Reagents:
-0/5 Blocking Agent 1w#en dry lyop#ili,ed pellet% stored at room temp in drawer6 w#en wet% stored in
-7/C in $8 bo9)4
-uclease-free water
0) ! using lyop#ili,ed 0/5 Blocking Agent from Agilent -yb :it% add 3// L of nuclease-free
water ! using lyop#ili,ed 0/5 Blocking Agent O8 found in kit 1Agilent p*n 30;;-37;04%
add 073/ L of nuclease-free water)
7) <ently (orte9) If pellet does not go into solution completely% #eat mi9 for =-3 minutes >
'?C) Centrifuge for 3 @ 0/ s)
Step ": Prep #yb sa$ples 1A 0 #our4
Reagents:
-0/5 Blocking Agent
-735 Bragmentation Buffer
-uclease-free water
-75 <+9 -ybridi,ation Buffer -I-$PM
0) +&uilibrate water bat# to ./C) 1Allow 0 #our for water bat# to warm up4
7) Bor eac# sample% add t#e following components to a separate /). mL tube
a) .// ng of labeled c$A
b) 3 L 0/5 Blocking Agent
c) 0 L 735 Bragmentation Buffer
') Bring final (olume of sample to "5 L by adding nuclease-free water
=) Corte9 well)
3) Incubate > ./C for e%actly '/ minutes to fragment $A) Immediately cool on ice for one
minute)
.) Add 73 L of 75 <+9 -yb Buffer -I-$PM to eac# tube to make 05 -yb Buffer) Cooling on
ice and t#e addition of -yb Buffer stops fragmentation reaction)
?) Mi9 by careful pipetting) Be sure to a(oid introducing bubbles6 do not (orte9)
;) Dpin for 0 minute at room temp at 0'%/// rpm to dri(e sample off tube walls E reduce
bubbles)
F) Place sample on ice E load onto array ADAP)
0/) Det water bat# to '?C immediately)
Step &: Asse$ble #yb c#a$ber 1A 0? #ours4
0) Load a clean gasket slide into #yb c#amber wit# t#e label 'acing (P and aligned wit# t#e
rectangular section of c#amber base) +nsure t#e gasket slide is flus# w* c#amber base E not
aGar) )*+E! if t#ere are unused gasket wells% place t#em at t#e far end opposite t#e bar code)
In t#e unused wells% maintain =/ L of 05 -yb Buffer)
7) Dlowly dispense =/ L of eac# sample into a new gasket well in a Hdrag E dispenseI manner)
Jo not let t#e pipette tip or t#e #yb solution touc# t#e gasket walls% w#ic# will increase t#e
risk of leaks)
') Dlowly place an array Hacti(e sideI down onto t#e Dure-yb gasket slide% so t#at t#e
HAgilentI @ labeled barcode is facing ,*-) and t#e numeric barcode is facing (P) Make
sure t#e sandwic#-pair is properly aligned) )*+E: w#en you lower microarray slide on top
of t#e Dure-yb gasket slide% make sure t#e two slides are parallel at all times) Jo not drop
array slide onto gasket% or you will increase t#e c#ance of sample mi9ing between wells)
=) Place t#e Dure-yb c#amber co(er onto t#e sandwic#ed slides E slide clamp assembly onto
bot# pieces)
3) -and-tig#ten t#e clamp onto t#e c#amber)
.) Certically rotate t#e assembled c#amber to wet t#e gasket E assess bubble mobility) If
necessary% gently tap assembly to mo(e stationary bubbles)
?) Place assembled slide c#amber in rotisserie in #yb o(en set to .3C) Be sure to balance
rotisserie of #yb o(en) Det rotation to 0/ rpm)
;) -ybridi,e at .3C for 0? #ours)
Step .: /icroarray -as# 1up to =3 minutes to allow water bat# to come to '?C from .3C4
)ote: "as# Buffer 7 needs to be warmed o(ernig#t in water bat#) Be sure to prep was# buffer t#e nig#t
before you plan to do t#e microarray was#)
Reagents:
-8riton 5-0/7 1only w#en was# buffers are 0
st
opened4
-<ene +9pression "as# Buffer 0
-<ene +9pression "as# Buffer 7
4A Add Triton X-102 to wash buffers
8#e addition of /)//3K 8riton 5-0/7 is optional% but #ig#ly recommended% as it reduces t#e possibility
of array was# artifacts) Add 8riton 5-0/7 to <ene +9pression was# buffer 0 and 7 bot# w#en t#e
cubitainer of was# buffers is first opened) Only needs to be added w#en opening was# buffers)
0) Carefully remo(e outer E inner caps from t#e cubitainer of was# buffer 0 and 7)
7) Lse a pipette to add 7 mL of pro(ided 0/K 8riton 5-0/7 into was# buffer into eac# was#
buffer)
') $eplace inner E outer gapes E mi9 t#e buffer carefully but t#oroug#ly by in(erting container
3-. times)
=) Carefully remo(e t#e outer E inner caps E install t#e spigot pro(ided w*was# buffer)
3) Label was# buffer bo9 to indicate addition of 8riton 5-0/7

8riton 5-0/7 can be added to smaller (olumes of was# buffer% as long as final dilution of 0/K 8riton 5-
0/7 is /)//3K in t#e was# buffer solution)
4B & 're!arm (ene )xpression *as Buffer 2
0) Bill = 3/ mL tubes wit# was# buffer 7 from t#e bo9 E place t#em in a '?C water bat#
o(ernig#t 1nig#t before was#4) 1It takes 7// mL of was# buffer 7 to fill dis#es4
D8OP #ere o(ernig#t to allow #ybrid,ation E warming of "as# Buffer 7
4C Wash microarray slides 10/ minutes4
+o be done )S,E t#e desktop #ood on nort# 0all
Dlide conditions
Step ,is# -as# B1''er +e$p +i$e
Jisassembly 0 <+ "as# Buffer 0 $oom temp
0
st
"as# 7
1on stir plate4
<+ "as# Buffer 0 $oom temp 0 minute
7
nd
"as# '
1on stir plate4
<+ "as# Buffer 7 +le(ated temp
1A'0C4
0 minute
0) $inse all staining dis#es% racks% and stir bars t#oroug#ly 13-. times4 wit# J+PC-treated
water) Jo not use any detergents% w#ic# can lea(e fluorescent residue)
7) Completely fill 0
st
slide-staining dis# wit# "as# Buffer 0 at room temp)
') Place a slide rack into 7
nd
slide-staining dis#) Add magnetic stir bar) Bill dis# w*enoug# "as#
Buffer 0 > room temp to co(er slide rack) Place dis# on magnetic stir plate)
=) Place empty dis# M' on stir plate E add magnetic stir bar) Jo not add prewarmed 1'?C4
"as# Buffer 7 yet)
3) $emo(e one #yb c#amber from incubate E record time% w#et#er bubbles formed% and if all
bubbles are mobile E rotating freely)
.) Jisassemble #yb c#amber!
a. Place c#amber on flat surface E loosen t#umbscrew
b. Dlide off clamp assembly E remo(e c#amber co(er
c. $emo(e array-gasket sandwic# from c#amber by grabbing slides from ends) :eep slide
numeric barcode facing LP as you transfer sandwic# to slide staining dis# M0)
d. "it#out letting go of slides% submerge gasket-sandwic# into dis# M0 containing was# buffer 0
?) "it# t#e sandwic# completely submerged in "as# Buffer 0% pry t#e sandwic# open from t#e
barcode end only!
a. Dlip one of t#e blunt ends of t#e forceps between t#e slides
b. <ently turn forceps up or down to separate slides
c. Let t#e gasket slide drop to t#e bottom of staining dis#
d. $emo(e microarray slide E place into slide rack in dis# M7 1Buffer 0 > room temp4)
Minimi,e e9posure to air) +ouc onl% te barcode portion of te microarra% or its edges,
;) $epeat steps 3-? for up to se(en additional slides in t#e group) Bor uniform was#ing% do up to
a ma9imum of ; slides)
F) "#en all slides are settled into slide rack% stir using setting = for 0 minute)
0/) "#ile stirring Buffer 0 into dis# M7% remo(e warm "as# Buffer 7 from water bat# and pour
into slide-staining dis# M') 1ote! t#e ele(ated temp of 7
nd
was# step is usually around '0C
due to cooling4
00) 8ransfer slide rack to slide-staining dis# M' 1w*was# buffer 7 > ele(ated temp4) Dtir using
setting = for 0 minute)
07) DLO"LN remo(e slide rack to minimi,e droplets on slide) 1D#ould take 3 @ 0/ seconds to
remo(e slide rack)4
0') Jiscard "as# Buffer 0 E 7 E refill dis#es if you are working wit# more t#an ; microarray
slides) If not% wait until after scanning to discard was# buffers)
0=) Put slides into slide #older wit# t#e Agilent barcode 'acing ,*-) in slide #older)
03) Immediately scan slide)
4 Wash !as"et slide to #re# for stri##in! 12 3 minutes4
Reagents: 1bot# located in 3/ mL tubes in desktop #ood
-"as# Buffer 7
-0//K +tO-
0) Dubmerge gasket slide into 3/ mL tube of "as# Buffer 7) Let sit for 0 minute and remo(e)
7) Dubmerge gasket slide into 3/ mL tube of 0//K +tO-) Let sit for 0 minute and remo(e
slowly to reduce droplets) 1D#ould take 3-0/ s to remo(e slide)4
') Let dry face up on a kimwipe in #ood)
Prod1ct in'or$ation
-its
<ene +9pression -ybridi,ation :it Agilent p*n 30;;-37=7
75 <+9 -ybridi,ation Buffer -I-$PM 17 (ials% 0)73 mL*(ial4
735 Bragmentation Buffer% 0// L
0/5 <ene +9pression Blocking Agent 1lyop#ili,ed pellet4
<ene +9pression "as# Buffer :it Agilent p*n 30;;-3'7?
<ene +9pression "as# Buffer 0% =L
<ene +9pression "as# Buffer 7% =L
8riton 5-0/7 10/K4% . (ials 10)'3 mL*(ial4
.ndividual products
0/5 Blocking Agent 1O=7F)//4 Agilent p*n 30;;-37;0
<ene +9pression "as# Buffer 0% =L 1O0?.)//4 Agilent p*n 30;;-3'73
<ene +9pression "as# Buffer 7% =L 1O0?.)//4 Agilent p*n 30;;-3'73
75 -I-$PM -ybridi,ation Buffer% 73 mL 1O=;F)//4 Agilent p*n 30F/-/=/'
735 Bragmentation Buffer% 0/ mL 1O70=)//4 Agilent p*n 30;3-3F?=
Dure-yb <asket slides 1; microarrays*slide4
0// gasket slides*roll 1O7??')//4 Agilent p*n <73'=-.//0.
7/ gasket slides*roll 1O./3)//4 Agilent p*n <73'=-.//03
3 gasket slides*roll 1O0?.)//4 Agilent p*n <73'=-.//0=