Edge lab, updated June 2011

Bioanalyzer Instructions
Adapted from Agilent RNA 6000 Nano Kit Guide G29389003!
Notes
"f t#ere$% a problem, u%e diagno%ti&% tab to get info to %end to Agilent'
Repla&e t#e %(ringe at t#e &#ip priming %tation ea&# time (ou %tart a ne) bo* of
2+ &#ip%' Agilent RNA 6000 Nano Kit: 5067-1511
,#en RNA ladder fir%t arri-e% .&ome% )it# 30/0 per -ial1, #eat denature tube for
2 min at 2034, &ool on i&e, and ali5uot into dail( u%e -ial%' Agilent rep
re&ommend% + /0, but onl( 1/0 i% needed per &#ip' 6tore -ial% at 8034 and t#a)
on i&e )#en read( to u%e'
Allo) reagent% to e5uilibrate to room temperature for 30 minute% before u%ing'
At least 30 minutes beore beginning
7ull out %ample% and ladder .ali5uoted into indi-idual u%e tube%1 from 8034
free8er9 :eep on i&e until u%e
7ull out t#e follo)ing reagent% from !34 fridge ; allo) to &ome to room temp
o 6000 Nano d(e .blue &ap1
o 6000 Nano mar:er .green &ap1
o 6000 Nano gel matri* .red &ap1 < only if needed; see below
!re"aring gel matri#$ =ou &an get eig#t6+/0 ali5uot% of gel matri* from one prep'
1' +00/0 Agilent RNA 6000 Nano gel matri* .red1 into %pin &olumn'
2' 6pin at !000 rpm for 10 min'
3' 6+ /0 elutant into eig#t 0'+ /0 tube%' 6tore at !34 for 1 mont#'
B%&'R% ()% 'N*+ , A--ing .ye
!' >orte* 6000 Nano d(e .blue1 for 10 %e& and 5ui&: %pin
+' Add 1 /0 d(e to t#e 6+/0 ali5uot of filtered gel
6' >orte* and %pin at 1!000 rpm for 10 min'
?Note@ "f (ou are running multiple &#ip% in 1 da(, (ou &an u%e remaining gelAd(e
matri* %olution' B#ere %#ould be enoug# gelAd(e matri* %olution in 1 ali5uot for at
lea%t 2 &#ip%'
)am"le "re" / loa-ing gel matri#
1' Cioanal(8er &an anal(8e a ma*imum of +00 ngA/0' "f ne&e%%ar(, dilute %ample
)it# RNa%efree )ater for a final -olume of 1 /0'
2' Deat denature %ample% at 2034 for 2 minute% t#en &ool on i&e'
3' ,#en loading t#e &#ip, al)a(% put t#e pipette tip to t#e bottom of t#e )ell to
di%pen%e li5uid'
!' 7ipette 9 /0 gel d(e mi* at t#e bottom of t#e )ell )it# t#e bold 0$ 7ut &#ip in
priming %tation, %et timer to 30 %e&, ma:e %ure plunger i% at 1 m0, and depre%% t#e
%(ringe until it lo&:% beneat# t#e lat&#' Bime )ait for 30 %e&' Relea%e plunger and
it )ill relea%e it%elf at lea%t until t#e 0'3 m0 mar:' ,ait for + %e& t#en pull plunger
up to t#e 1 m0 mar:'
Edge lab, updated June 2011
+' Epen priming %tation and pipette 9 0 gel d(e mi* into ea&# remaining G )ell'
*oa-ing mar1er2 la--er2 an- sam"les
1' 7ipette + /0 RNA 6000 Nano mar:er .green1 into )ell mar:ed )it# ladder
%(mbol and ea&# of t#e 12 %ample )ell' .All )ell% NEB mar:ed G1
2' 7ipette 1 /0 ladder into )ell mar:ed )it# a ladder %(mbol'
3' 7ipette 1 /0 %ample% into 12 %ample )ell%'
!' FE NEB run empt( )ell% on a &#ip' Al)a(% fill e-er( )ell )it# a %ample'
+' >orte* on 2!00 rpm for 60 %e& .it %#ould %top b( it%elf1'
6' 7la&e &#ip in bioanal(8er, &lo%e lid, load %oft)are'
2' 6ele&t appropriate a%%a( from Assay menu .Electrophoresis, RNA1, %ele&t
Eukaryotic Total RNA Nano, t#en &li&: t#e Start button' Gill out %ample name%'
Run )ill ta:e H30 min'
?Note < if (ou are running multiple &#ip%, a-oid allo)ing &#ip% to %it around for
more t#an + minute% before running on t#e bioanal(8er' ,ait until t#e &omputer
nearl( fini%#e% one &#ip before %tarting t#e prep on t#e ne*t &#ip'
.ata )a3ing
Cioanal(8er automati&all( %a-e% file% under t#e 4 dri-e a% an '*ad file, )#i&# i%
onl( readable b( bioanal8(er %oft)are' Gile dire&tor(@ I( 4omputer 4 dri-e
7rogram Gile% Agilent 2100 Cioanal(8er 2100 e*pert data' Jnder
t#e Fata folder, file% are organi8ed b( data
Bo print data a% 7FG, &li&: K7rint'L B#i% )ill allo) (ou to %a-e t#e data a% a 7FG
file in t#e folder KCioanal(8er 7FG%L .file dire&tor(@ I( 4omputer Fata .F
dri-e1 Cioanal(8er 7FG%, )#ere t#e( are organi8ed b( date'1 B#ere i% a
%#ort&ut on t#e de%:top to t#e Cioanal(8er 7FG% folder a% )ell'
En&e (ou open t#e 7FG, (ou &an print' B#e 7FG i% 18 page% long, %o be %ure to
print onl( t#e 1
%t
%ummar( page, unle%% (ou need more' Ce %ure to %a-e a &op( of
t#e 7FG under t#e appropriate I dri-e folder'
4leanu"
1' 4lean tip of %(ringe after e-er( u%e )it# i%opropanol, )ipe dr( )it# Kim )ipe
to pre-ent &logging' Repla&e if gel doe% not run e-enl('
2' After e-er( run, u%e &leaner &#ip% .1 )it# RNa%e A)a(, 1 RNa%e free )ater1 to
&lean ele&trode pin %et' Ju%t allo) bioanal(8er to be on for 1 min ea&#'
6oft)are doe% not #a-e to be running' 4#ip% %#ould be filled )it# 3+0/0
RNa%e A)a( or )ater' ,ait 10 %e&ond% for )ater to e-aporate from ele&trode%
before &lo%ing lid'
3' En&e a )ee:, &lean probe%' 7our RNa%efree )ater onto toot#bru%# and gentl(
)ipe' 0ea-e t#e lid of t#e bioanal(8er open for 10 %e&ond% to allo) )ater to
e-aporate from probe%'