Agilent Hyb Protocol 5.5.2011

8/11/2019 Agilent Hyb Protocol 5.5.2011

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Edge Lab Agilent Hybridization Protocol – 5/5/11Adapted 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// µL of nuclease-free

water ! using lyop#ili,ed 0/5 Blocking Agent O8 found in kit 1Agilent p*n 30;;-37;04%add 073/ µL 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 1 0 #our4

Reagents:

-0/5 Blocking Agent-735 ragmentation 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) or eac# sample% add t#e following components to a separate /). mL tubea) .// ng of labeled c$A

b) 3 µL 0/5 Blocking Agent

c) 0 µL 735 ragmentation Buffer

') Bring final (olume of sample to "5 L by adding nuclease-free water

=) orte9 well)

3) Incubate > ./°C for e%actly '/ minutes to fragment $A) Immediately cool on ice for oneminute)

.) Add 73 µL 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 1 0? #ours4



0) Load a clean gasket slide into #yb c#amber wit# t#e label 'acing (P and aligned wit# t#erectangular section of c#amber base) +nsure t#e gasket slide is flus# w* c#amber base E notaGar) )*+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 =/ µL of 05 yb Buffer)

7) Dlowly dispense =/ µL of eac# sample into a new gasket well in a Hdrag E dispense manner)

Jo not let t#e pipette tip or t#e #yb solution touc# t#e gasket walls% w#ic# will increase t#erisk of leaks)

') Dlowly place an array Hacti(e side down onto t#e Dureyb gasket slide% so t#at t#eHAgilent @ labeled barcode is facing ,*-) and t#e numeric barcode is facing (P) Makesure t#e sandwic#-pair is properly aligned) )*+E: w#en you lower microarray slide on topof t#e Dureyb gasket slide% make sure t#e two slides are parallel at all times) Jo not droparray slide onto gasket% or you will increase t#e c#ance of sample mi9ing between wells)

=) Place t#e Dureyb 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).) ertically 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 .3°C) Be sure to balance

rotisserie of #yb o(en) Det rotation to 0/ rpm);) ybridi,e at .3°C for 0? #ours)

Step .: icroarray -as# 1up to =3 minutes to allow water bat# to come to '?°C from .3°C4

)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 0st 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 possibilityof array was# artifacts) Add 8riton 5-0/7 to <ene +9pression was# buffer 0 and 7 bot# w#en t#ecubitainer 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) se 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) ill = 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# B''er +e$p +i$e

Jisassembly 0 <+ "as# Buffer 0 $oom temp

0st "as# 71on stir plate4

<+ "as# Buffer 0 $oom temp 0 minute

7nd "as# '1on stir plate4

<+ "as# Buffer 7 +le(ated temp

1'0°C4

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 0st slide-staining dis# wit# "as# Buffer 0 at room temp)') Place a slide rack into 7nd slide-staining dis#) Add magnetic stir bar) ill dis# w*enoug# "as#

Buffer 0 > room temp to co(er slide rack) Place dis# on magnetic stir plate)

=) Place empty dis# ' 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#umbscrewb. 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 slidenumeric barcode facing P as you transfer sandwic# to slide staining dis# 0)

d. "it#out letting go of slides% submerge gasket-sandwic# into dis# 0 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 slidesb. <ently turn forceps up or down to separate slidesc. 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# 7 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) or 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# 7% remo(e warm "as# Buffer 7 from water bat# and pour

into slide-staining dis# ') 1ote! t#e ele(ated temp of 7nd was# step is usually around '0°C

due to cooling400) 8ransfer slide rack to slide-staining dis# ' 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)40') 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)

Prodct in'or$ation

its

<ene +9pression ybridi,ation :it Agilent p*n 30;;-37=775 <+9 ybridi,ation Buffer I-$PM 17 (ials% 0)73 mL*(ial4

735 ragmentation Buffer% 0// µL

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% =L8riton 5-0/7 10/K4% . (ials 10)'3 mL*(ial4

ndividual products

0/5 Blocking Agent 1=7F)//4 Agilent p*n 30;;-37;0

<ene +9pression "as# Buffer 0% =L 10?.)//4 Agilent p*n 30;;-3'73

<ene +9pression "as# Buffer 7% =L 10?.)//4 Agilent p*n 30;;-3'73

75 I-$PM ybridi,ation Buffer% 73 mL 1=;F)//4 Agilent p*n 30F/-/=/'

735 ragmentation Buffer% 0/ mL 170=)//4 Agilent p*n 30;3-3F?=

Dureyb <asket slides 1; microarrays*slide40// gasket slides*roll 17??')//4 Agilent p*n <73'=-.//0.7/ gasket slides*roll 1./3)//4 Agilent p*n <73'=-.//033 gasket slides*roll 10?.)//4 Agilent p*n <73'=-.//0=

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Agilent Hyb Protocol 5.5.2011