BIOC7001 – Advanced DNA Techniques 2011_FINAL

scmb.uq.edu.au

School of Chemistry & Molecular Biosciences, Faculty of Science

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#,$-.//0(Advanced DNA Technology Semester 2, 2011

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Ensure that you have completed the on-line Safety Declaration and read the associated Occupational Health and Safety in the Laboratory (Undergraduate Student Edition) Safety

Guideline before commencing practical work. !!

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Week Day (Lab sessions are 10 am – 5 pm unless otherwise stated)

Mon Tues Weds Thurs Fri

1 (25/7)

2 (1/8) Introductory

Practical

3 (8/8)

4 (15/8)

5 (22/8) Module 1 Practical

Module 1 Practical

Module 1 Practical

(4 pm finish)

Module 1 Practical





Module 3 Practical

Module 3 Practical

10 (3/10)

11 (10/10)

12 (17/10)

13 (24/10)

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5’-AGCTAGCTAGCGCTAGCGAGCTAGCGATCGAGCTAGCGATCGAGCTAGC-3’

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3’-TCGATCGATCGCGATCGCTCGATCGCTAGCTCGATCGCTAGCTCGATCG-5’

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B'B'D E3*/,8$26$>0.+3F$N@$!8$(/'0!(=+88.2.(+-./'!/2!%$8-%.(-./'!$'Q)#$8!.8!7+8$0!/'!-@$!'+-,%$!/2!-@$!$'08!/2!

-@$!(=$+*$0!:EA!4%/0,($05!:.22$%$'-!%$8-%.(-./'!$'Q)#$8!(+'!(=$+*$!7/-@!8-%+'08!+-!-@$!8+#$!4/.'-!=$+*.'&!+!p7=,'-q!$'0!/%!#+)!(=$+*$!$+(@!8-%+'0!+-!4/8.-./'8!/228$-!2%/#!$+(@!/-@$%!%$8,=-.'&!.'!/'$!8-%+'0!/2!:EA!7$.'&!=/'&$%!-@+'!-@$!/-@$%!&.*.'&!+'!p/*$%@+'&q!/%!p8-.(;)!$'0q5!9/%!%$8-%.(-./'!$'Q)#$8!-@+-!&.*$!+'!/*$%@+'&!2/%!0.22$%$'-!$'Q)#$8!+!0.22$%$'-!8-%+'0!(+'!7$!-@$!=/'&$%!/'$L!&.*.'&!$.-@$%!+!Ob!/%!Mb!/*$%@+'&5!"D+#4=$8!/2!-@$!0.22$%$'-!-)4$8!/2!(=$+*+&$!8.-$8!+%$!8@/['!7$=/[5!

!

U)#!>V!T7=,'-U!Ob<ANANIMb!MbINANA<Ob!

Ob<ANMb!!!!!!!!!!!!!!ObANIMb!MbINAOb!!!!!!!!!!!!!!MbNA<Ob!

U)#!>C!TOb!/*$%@+'&U!Ob<AANNIMb!MbINNAA<Ob!

Ob<Mb!!!!!!!!!!!!!!ObAANNIMb!MbINNAAOb!!!!!!!!!!!!!!Mb<Ob!

7()!C!TMb!/*$%@+'&U!Ob<A<INIMb!MbINI<A<Ob!

Ob<A<INMb!!!!!!!!!!!!!!ObIMb!MbIOb!!!!!!!!!!!!!!MbNI<A<Ob!

!Z'($!-@$!:EA!@+8!7$$'!(=$+*$0!.-!(+'!7$!g/.'$0!T=.&+-$0U!-/!+'/-@$%!4.$($!/2!:EA!-@+-!

@+8!+!(/#4=$#$'-+%)!$'0!,8.'&!-@$!$'Q)#$!:EA!=.&+8$5!`,.-$!/7*./,8=)!.2!)/,!@+*$!+!7=,'-!$'0!.-!(+'!7$!=.&+-$0!-/!+')!/-@$%!7=,'-K$'0$0!4.$($!/2!:EA5!9/%!-@$!8-.(;)!$'08!-@$!-[/!4.$($8!/2!:EA!@+*$!-/!@+*$!(/#4=$#$'-+%)!/*$%@+'&8!-/!+==/[!=.&+-./'!-/!/((,%[email protected]!/2-$'!#$+'8!-@+-!7/-@!4.$($8!/2!:EA!@+*$!7$$'!(=$+*$0!,8.'&!-@$!8+#$!$'Q)#$!7,-!-@$%$!+%$!.'8-+'($8![@$%$!-[/!0.22$%$'-!$'Q)#$8L![.-@!0.22$%$'-!%$(/&'.-./'!8.-$8L!(+'!&.*$!(/#4=$#$'-+%)!8-.(;)!$'085!

!1.2 The polymerase chain reaction (PCR)

N@$!0$*$=/4#$'-!/2!BI>!%$*/=,-./'.8$0!/,%!+7.=.-)!-/!#+'.4,=+-$!+'0!.8/=+-$!:[email protected]!-$(@'.3,$!+==/[8!%$8$+%(@$%8!-/!p(/4)q!/%!p+#4=.2)q!*$%)!84$(.2.(!-+%&$-!8$3,$'($8!/2!:EA5!?$!(+'!-@$'!-+;$!-@$8$!+#4=.2.$0!8$3,$'($8!T+#4=.(/'8U!+'0!+'+=)8$!-@$#!,8.'&!&$=!$=$(-%/4@/%$8.8L!(=$+*$!-@$#!,8.'&!%$8-%.(-./'!$'Q)#$8!/%!(=/'$!-@$#!.'-/!+!8,.-+7=$!4=+8#.0!*$(-/%5!

N@$!BI>!-$(@'.3,$!+44%/4%.+-$8!-@$!+7.=.-)!/2!:EAK0$4$'0$'-!:EA!4/=)#$%+8$8!-/!,8$!+!8.'&=$!8-%+'0!/2!:EA!+8!+!-$#4=+-$!+'0!(+-+=)8$!-@$!+00.-./'!/2!(/#4=$#$'-+%)!7+8$8!-/!8)'-@$8.8$!-@$!(/#4=$#$'-+%)!8-%+'05!S/P(;B(;@/(GF/:<D<:<;P(BD(8L%(<G(;@/(D9:;(;@9;(ZT"(

FB=P>/09G/G(:9K(BK=P(9CC(K/J(KE:=/B;<C/G(;B(;@/(/HFBG/C(Qe(/KC(BD(9K(/H<G;<KO(F</:/(BD(ZT"[email protected]!#$+'8!+!:EA!4/=)#$%+8$!(+''/-!(/4)!+')!4.$($!/2!8.'&=$K8-%+'0$0!:EAL!.-!#,8-!=/(+-$!+!%$&./'![@$%$!-@$%$!.8!+!8@/%-!8$(-./'!/2!0/,7=$K8-%+'0$0!:EA!2%/#![@.(@!.8!+0g+($'-!-/!+!8.'&=$K8-%+'0$0!%$&./'5!

C-!.8!/'=)![@$%[email protected]!+%%+'&$#$'-!/((,%8!-@+-!-@$!:EA!4/=)#$%+8$!(+'!7.'0!+'0!8-+%-!$D-$'0.'&!-@$!,44$%!8-%+'05!

BI>!.8!7+8$0!/'!4%/*.0.'&!-[/L!8@/%-!0/,7=$K8-%+'0$0!%$&./'8!F!/'$!+-!$.-@$%!$'0!/2!-@$!-+%&$-!8$(-./'!/2!:EA5!?$!(+'!0/[email protected]!7)!,8.'&!8@/%-L!+%-.2.(.+==)!8)'-@$8.8$0L!8.'&=$K

5’-AGCTAGCTA-3’

|||||||||

3’-TCGATCGATCGCGATCGCTCGATCGCTAGCTCGATCGCTAGCTCGATCG-5’

!"#$%&'()'!"%&*$%+,'*%"-.-/'

#$%&'(()"*+,-./0+"12*"30/4.56578"9())" \"

!

8-%+'0$0!4.$($8!/2!:EA!;'/['!+8!/=.&/',(=$/-.0$!4%.#$%85!9/%!+!8-+'0+%0!BI>![$!%$3,.%$!-[/!4%.#$%8!F!/'$!-@+-!.8!(/#4=$#$'-+%)!-/!-@$!7/--/#!8-%+'0!T8$$!+7/*$U!+'0!/'$!-@+-!.8!(/#4=$#$'-+%)!-/!-@$!,44$%!8-%+'0!T8$$!7$=/[U5!

!N@$!2.%8-!8-$4!.'!+')!BI>!.8!-/!8$4+%+-$!-@$!0/,7=$K8-%+'0$0!:EA![$![.8@!-/!+#4=.2)L!

;'/['!+8!-$#4=+-$!:[email protected]!.8!0/'$!7)!@$+-.'&!-/!+%/,'0!^Y{I!+'0!.8!-$%#$0!C/K9;E09;<BK5!N@$'!.'!-@$!4%$8$'($!/2!/,%!/=.&/',(=$/-.0$!4%.#$%8![$!%$0,($!-@$!-$#4$%+-,%$!F!-@$!-$#4$%+-,%$!=/[$%!-/!.-!-$%#$0!-@$!9KK/9=<KO!-$#4$%+-,%$!+'0!.8!0$-$%#.'$0!7)!-@$!=$'&-@!+'0!7+8$!(/#4/8.-./'!/2!-@$!4%.#$%85!A8!+!%,=$K/2K-@,#7!+!-$#4$%+-,%$!O{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email protected]!$'Q)#$![+8!.8/=+-$0!2%/#!-@$!-@$%#/4@.=.(!+%(@+$+!PQ*$8.1!(V.(43).1!+'0!@+8!-@$!+0*+'-+&$!/2!7$.'&!8-+7=$!+-!@.&@!-$#4$%+-,%[email protected]!@+8!-[/!+0*+'-+&$8!F!G5!N@$!$'Q)#$!.8!+7=$!-/!8,%*.*$!-@$!@.&@!-$#4$%+-,%$8!/2!-@$!0$'+-,%+-./'!8-$4o!H5!N@$!-$#4$%+-,%$!/4-.#,#!2/%!-@$!$'Q)#$!.8!PH{I!#$+'.'&!-@+-!$D-$'8./'!(+'!/((,%!+-!+!-$#4$%+-,%$!+-![@.(@!-@$!(@+'($!/2!-@$!4%.#$%8!7.'0.'&!.'!+!'/'K84$(.2.(!#+''$%!.8!&%$+-=)!%$0,($05!N@$!-.#$!+==/[$0!2/%!$D-$'8./'!.8!-.&@-=)!(/'-%/==$0!F!!$'/,&@!-.#$!.8!&.*$'!2/%!-@$!:EA!4/=)#$%+8$!-/!p(/4)q!-@$!$'-.%$!=$'&-@!/2!-@$!-+%&$-!8$3,$'($!7,-!-@$!-.#$!.8!%$8-%.(-$0!-/!4%$*$'-!-@$!:EA!4/=)#$%+8$!2%/#!2%,.-=$88=)!(/4).'&!#/%$!:EA!-@+'!.8!'$($88+%)5!!

N/!&%$+-=)!+#4=.2)!-@$!-+%&$-!8$3,$'($!-@$!()(=$!/2!0$'+-,%+-./'L!+''$+=.'&!+'0!$D-$'8./'!.8!%$4$+-$0!HOKYW!-.#$8!0$4$'0$'-!/'!-@$!4+%-.(,=+%!4%/-/(/=5!A2-$%!$+(@!%/,'0!'/-!/'=)!(+'!-@$!4%.#$%8!7.'0!-@$!/%.&.'+=!-$#4=+-$!:EA!7,-!-@$)!(+'!+=8/!7.'0!-@$!'$[=)!8)'-@$8.8$0!8-%+'085!A2-$%!8$*$%+=!()(=$8!-@$!'$[=)!8)'-@$8.8$0!8-%+'08!+%$!2+%!#/%$!',#$%/,8!-@+'!-@$!/%.&.'+=!-$#4=+-$!:EA!+'0!-@$!(/4).'&!/2!-@$8$!0/#.'+-$8!T8$$!0.+&%+#U!! !

5’-AGCTAGCTAGCGCTAGCGAGCTAGCGATCGAGCTAGCGATCGAGCTAGC-3’

|||||||

3’-TCGATCG-5’

!"#$%&'()'!"%&*$%+,'*%"-.-/'

)(" #$%&'(()"*+,-./0+"12*"30/4.56578"9())"

!

1.3 Recombinant Plasmid Vectors B=+8#.08!+%$!'+-,%+==)!/((,%%.'&!(.%(,=+%!:EA!#/=$(,=$8!-@+-!(+'!7$!2/,'0!.'!+!',#7$%!

/2!7+(-$%.+=!84$(.$85!"+(@!4=+8#.0!.8!+7=$!-/!7$!%$4=.(+-$0!T(/4.$0o!0,4=.(+-$0U!7)!-@$!:EA!%$4=.(+-.'&!$'Q)#$8!/2!-@$!7+(-$%.,#[email protected]!#$+'8!-@$)!(+'!7$!#+.'-+.'$0!+'0!4+88$0!/'!-/!-@$!7+(-$%.,#b8!4%/&$')5!

>$(/#7.'+'-!4=+8#.0!*$(-/%8!+%$!7+8$0!/'!-@$!8+#$!4%.'(.4=$8!+8!-@$.%!'+-,%+==)!/((,%%.'&!(/,8.'85!:,%.'&!-@$!0$*$=/4#$'-!/2!-@$8$!+%-.2.(.+=!*$(-/%8!-@$!%$8,=-.'&!4=+8#.08!+%$!8-%$+#=.'$0!F!-@$)!@+*$!-@$!#.'.#+=!',#7$%!/2!7+8$!4+.%8!%$3,.%$0!-/!$'(/0$!+==!/2!-@$!;$)!2$+-,%$8!+'0!8/!+%$!/2-$'!#,(@!8#+==$%!-@+'!'+-,%+==)!/((,%%.'&!4=+8#.085!

A=8/!-@$!%$(/#7.'+'-!*$(-/%8!@+*$!+00.-./'+=!2$+-,%$8!-@+-!.'(%$+8$!-@$.%!,-.=.-)!2/%!-@$!%$8$+%(@!8(.$'-.8-5!J$=/[!.8!+!=.8-!/2!&$'$%+=!2$+-,%$8!2/,'0!.'!#+')!(/##$%(.+==)!+*+.=+7=$!4=+8#.0!*$(-/%8!+'0!-@$.%!2,'(-./'5!!

!*/9;E0/( *EK:;<BK(Z%.&.'!/2!%$4=.(+-./'!

[email protected]!.8!+!84$(.2.(!8$3,$'($!/2!:EA!0$2.'.'&![@$%$!:EA!4/=)#$%+8$!(+'!8-+%-!%$4=.(+-.'&!-@$!4=+8#[email protected]!2$+-,%$!.8!;$)!-/!-@$!+7.=.-)!/2!-@$!7+(-$%.,#!-/!#+.'-+.'!-@$!4=+8#.0!+'0!4+88!/'!(/4.$8!-/!.-8!4%/&$')[email protected]!2$+-,%$!#$+'8!-@+-![.-@!(+%$2,=!#+.'-$'+'($!/2!-@$!-%+'82/%#$0!7+(-$%.+=!8-%+.'!-@$!4=+8#.0!(+'!7$!;$4-!.'!4$%4$-,.-)5!!

1$=$(-.*$!#+%;$%! N%+'82/%#+-./'!4%/-/(/=8!(+''/-!&,+%+'-$$!-@+-!$*$%)!.'0.*.0,+=!7+(-$%.,#![.==!-+;$!,4!4=+8#.05!N/!+==/[!-@$!%$8$+%(@$%!-/!8$=$(-!-@/8$!7+(-$%.+!-@+-!@+*$!7$$'!-%+'82/%#$0!-@$!4=+8#.0!*$(-/%8!(/'-+.'!/'$!/%!#/%$!&$'$8!$'(/0.'&!+!8$=$(-.*$!#+%;$%L!/2-$'!+!&$'$!$'(/0.'&!+'-.7./-.(!%$8.8-+'([email protected]!#$+'8!-@+-!2/==/[.'&!-%+'82/%#+-./'!-@$!7+(-$%.+!(+'!7$!&%/['!.'!-@$!4%$8$'($!/2!-@$!+44%/4%.+-$!+'-.7./-.(!F!-%+'82/%#$0!($==8![.==!8,%*.*$!+'0!&%/[o!'/'K-%+'82/%#$0!($==8![.==!4$%.8@5!

X,=-.4=$!(=/'.'&!8.-$!TXI1U!

[email protected]!.8!+!8$%.$8!/2!%$8-%.(-./'!$'Q)#$!%$(/&'.-./'!8.-$8!+%%+'&$0!(/'8$(,-.*$=)!.'!+!8#+==!%$&./'!/2!-@$!4=+8#.0!*$(-/%5!C'!&$'$%+=!$+(@!/2!-@$!%$8-%.(-./'!$'Q)#$8!8.-$8![.-@.'!+!XI1!+%$!,'.3,$!F!-@$%$!+%$!'/!/-@$%!8.-$8!2/%!-@$!%$8-%.(-./'!$'Q)#$!+')[@$%$!$=8$!.'!-@$!4=+8#.0!*$(-/%5!

6+(!f!&$'$! [email protected]!2$+-,%$!.8!2/,'0!.'!#+')!7,-!'/-!+==!4=+8#.0!*$(-/%[email protected]!&$'$!$'(/0$8!2/%!+!|K&+=+(-/8.0+8$!$'Q)#[email protected]!$'Q)#$!.8!'/%#+==)!,-.=.8$0!.'!-@$!7%$+;0/['!/2!=+(-/8$!7,-!(+'!+=8/!7%$+;0/['!/-@$%L!+%-.2.(.+=!8,78-%+-$85!N%+'82/%#$0!7+(-$%.+!(+'!7$!&%/['!.'!-@$!4%$8$'($!/2!C8/4%/4)=!|K:KGK-@./&+=+(-/4)%+'/8.0$!TCBN<U!+'0!OK7%/#/KYK(@=/%/KMK.'0/=)=K7$-+K:K&+=+(-/4)%+'/8.0$!TzK<+=U5!CBN<!.'0,($8!-@$!6+(f!&$'$!=$+0.'&!-/!-@$!8)'-@$8.8!/2!-@$!|K&+=+(-/8.0+8$!$'Q)#$5!N@$!zK<+=!.8!+!8,78-%+-$!2/%!-@$!|K&+=+(-/8.0+8$!$'Q)#$![@.(@![@$'!7%/;$'!0/['!).$=08!+!7=,$!(/#4/,'05!N@$!XI1!/2!-@$!4=+8#.0!*$(-/%!.8!8.-,+-$0![.-@.'!-@$!6+(f!&$'$!F![@$'!+!:EA!2%+&#$'-!.8!(=/'$0!.'-/!-@[email protected]!.'-$%%,4-8!-@$!6+(f!&$'$!#+;.'&!.-!.'/4$%+-.*[email protected]!.8!-@$!7+8.8!/2!7=,$K[@.-$!8(%$$'.'&!F!($==8![@+-!(/'-+.'!+!4=+8#.0!*$(-/%![@.(@!@+8!+!(=/'$0!.'8$%-![.==!%$#+.'![@.-$![@$'!&%/['!.'!-@$!4%$8$'($!/2!CBN<!+'0!zK<+=5!

!

!"#$%&'()'!"%&*$%+,'*%"-.-/'

#$%&'(()"*+,-./0+"12*"30/4.56578"9())" ))"

!

B'G'B <7/316*70$367+&H26:73*2+$N/!,-.=.8$!-@$8$!%$(/#7.'+'-!4=+8#.08!-@$)!#,8-!7$!.'-%/0,($0!.'-/!+!8,.-+7=$!@/8-5!9/%!

[email protected]!#/0,[email protected]!@/8-![.==!7$!+!8-%+.'!/2!-@$!=+7/%+-/%)![/%;@/%8$!U9!)#'35!N@$!4%/($88!/2!.'-%/0,(.'&!'$[!&$'$-.(!#+-$%.+=!.'-/!U9!)#'3!(+'!-%+'82/%#!.-8!4@$'/-)4$!T.-8!/78$%*+7=$!(@+%+(-$%.8-.(8U!+'0!8/!-@$!-$%#!,8$0!2/%!-@$!4%/-/(/=!/2!.'-%/0,(.'&!+!4=+8#.0!.'-/!.-8!7+(-$%.+=!@/8-!.8!;09KGDB0>9;<BK!T-@$!*$(-/%!.8!,8$0!-/!-%+'82/%#!-@$!7+(-$%.,#U5!

i'0$%!'/%#+=!(/'0.-./'8!U9!)#'3![/,=0!'/-!-+;$!,4!$D-%+($==,=+%!:EA5!N@,8L!-@$!7+(-$%.+!2.%8-!@+*$!-/!7$!-%$+-$0!-/!$'+7=$!-@$#!-/!0/[email protected]!F!7+(-$%.+!-%$+-$0!.'[email protected]![+)!+%$!8+.0!-/!7$!:B>F/;/K;5!

!

B'G'D ;IJK?A$J7&8$C'[email protected]!#/0,=$!)/,![.==!7$!,8.'&!-@$!B%/#$&+!4=+8#.0!*$(-/%!4<"XKN!"+8)[email protected]!*$(-/%!

@+8!+==!/2!-@$!2$+-,%$8!#$'-./'$0!+7/*$!F!+!XI1L!+!8$=$(-.*$!#+%;$%!T+#4.(.==.'!%$8.8-+'($UL!+'!/%.&.'!/2!%$4=.(+-./'!+'0!-@$!6+(!f!&$'$5!!

N@$!#+g/%!0.22$%$'($!7$-[$$'[email protected]!*$(-/%!+'0!/-@$%!.8!-@+-!.-!@+8!7$$'!=.'$+%.Q$0!7)!(=$+*+&$![.-@.'!-@$!XI15!N@$!/'!$+(@!$D4/8$0!$'0!+!N!',(=$/-.0$!@+8!7$$'!+00$0!-/!-@$!$D4/8$0!Mb!$'0!/2!/'$!8-%+'0!F!(%$+-.'&!+!/'$!7+8$L!Mb!/*$%@+'&5!!

!

!A!4$(,=.+%!3,.%;!/2!P(V!4/=)#$%+8$!.8!-@+-!0,%.'&!-@$!+#4=.2.(+-./'!/2!+')!-+%&$-!:EA!

-@$!%$8,=-+'-!4%/0,(-!.8!#+0$![.-@!+!/'$!7+8$L!Mb!/*$%@+'&!/2!+'!A!7+8$5!!

5’--AGCTAGCTAGCGCTAGCGAGCTAGCGATCGAGCTAGCGATCGAGCTAGCA-3’

|||||||||||||||||||||||||||||||||||||||||||||||||

3’-ATCGATCGATCGCGATCGCTCGATCGCTAGCTCGATCGCTAGCTCGATCG--5’ [email protected]!/*$%@+'&!.8!(/#4=$#$'-+%)!-/!-@$!N!/*$%@+'&!/2!-@$!4<"XKN!"+8)!*$(-/%!+'0!8/!

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4.C. Screening Transformants for Inserts

Successful cloning of an insert into the pGEM®-T or pGEM®-T Easy Vectorinterrupts the coding sequence of !-galactosidase; recombinant clones can beidentified by color screening on indicator plates. However, the characteristics ofthe PCR products cloned into the vectors can significantly affect the ratio ofblue:white colonies obtained. Usually clones containing PCR products producewhite colonies, but blue colonies can result from PCR fragments that are clonedin-frame with the lacZ gene. Such fragments are usually a multiple of 3 basepairs long (including the 3´-A overhangs) and do not contain in-frame stopcodons. There have been reports of DNA fragments up to 2kb that have beencloned in-frame and have produced blue colonies. Even if your PCR product isnot a multiple of 3 bases long, the amplification process can introducemutations (deletions or point mutations) that may result in blue colonies.

The Control Insert DNA supplied with the pGEM®-T and pGEM®-T EasySystems is a 542bp fragment from pGEM®-luc Vector DNA (Cat.# E1541). Thissequence has been mutated to contain multiple stop codons in all six readingframes, which ensures a low background of blue colonies for the controlreaction. Results obtained with the Control Insert DNA may not berepresentative of those achieved with your PCR product.

5. pGEM®-T and pGEM®-T Easy Vector Sequences, Multi-Cloning Sites andCircle Maps

5.A. Sequence and Multi-Cloning Site of the pGEM®-T Vector

The pGEM®-T Vector is derived from the pGEM®-5Zf(+) Vector (GenBank®

Accession No. X65308). The pGEM®-T Vector was created by linearizing thepGEM®-5Zf(+) Vector with EcoRV at base 51 and adding a T to both 3´-ends.The EcoRV site will not be recovered upon ligation of the vector and insert.

Promega Corporation ! 2800 Woods Hollow Road ! Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526 ! Phone 608-274-4330 ! Fax 608-277-2516 ! www.promega.comPart# TM042 Printed in USA.Page 8 Revised 12/10

GGAGA GCTCC CAACG CGTTG GATGC ATAGC TTGAG TATTC TATAG TGTCA CCTAA AT . . . 3!CCTCT CGAGG GTTGC GCAAC CTACG TATCG AACTC ATAAG ATATC ACAGT GGATT TA . . . 5!

CCATG GCCGC GGGATT3! ATCAC TAGTG CGGCC GCCTG CAGGT CGACC ATATG GGTAC CGGCG CCCTA 3!TTAGTG ATCAC GCCGG CGGAC GTCCA GCTGG TATAC

5! . . . TGTAA TACGA CTCAC TATAG GGCGA ATTGG GCCCG ACGTC GCATG CTCCC GGCCG 3! . . . ACATT ATGCT GAGTG ATATC CCGCT TAACC CGGGC TGCAG CGTAC GAGGG CCGGC

SP6 Promoter

SP6 Transcription Start

SacI BstXI NsiI

T7 Transcription Start

T7 PromoterApaI AatII SphI BstZI

BstZI

(cloned insert)SacIINcoI PstINotI NdeISpeI SalI

0357

MA

06_2

A

Figure 1. The promoter and multiple cloning sequence of the pGEM®-T Vector. The top strand corresponds to the RNA synthesized by T7 RNA polymerase. Thebottom strand corresponds to the RNA synthesized by SP6 RNA polymerase.

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1.9 Appendices

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5.D. pGEM®-T Easy Vector Map and Sequence Reference Points

pGEM®-T Easy Vector sequence reference points:

T7 RNA polymerase transcription initiation site 1multiple cloning region 10–128SP6 RNA polymerase promoter (–17 to +3) 139–158SP6 RNA polymerase transcription initiation site 141pUC/M13 Reverse Sequencing Primer binding site 176–197lacZ start codon 180lac operator 200–216!-lactamase coding region 1337–2197phage f1 region 2380–2835lac operon sequences 2836–2996, 166–395pUC/M13 Forward Sequencing Primer binding site 2949–2972T7 RNA polymerase promoter (–17 to +3) 2999–3

Note: Inserts can be sequenced using the SP6 Promoter Primer (Cat.# Q5011), T7Promoter Primer (Cat.# Q5021), pUC/M13 Forward Primer (Cat.# Q5601), orpUC/M13 Reverse Primer (Cat.# Q5421).

Note: A single digest with BstZI (Cat.# R6881), EcoRI (Cat.# R6011) or NotI (Cat.#R6431) will release inserts cloned into the pGEM®-T Easy Vector. Double digests canalso be used to release inserts.

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9/%!-@$!$(/'/#.(+==)!.#4/%-+'-!($%$+=8!T%.($L!#+.Q$L!/+-8!+'0![@$+-UL!$22.(.$'-!-%+'82/%#+-./'![+8!#/%$!0.22.(,=-!-/!+(@.$*$5!c/[$*$%L!#/8-!/2!-@$8$!84$(.$8!(+'!'/[!7$!%/,-.'$=)!-%+'82/%#$0!7)!L9!4.8*T()3*,1C!T$5&5!c.$.!*4!('9L!G^^YU!+'0d/%!4+%-.(=$!7/#7+%0#$'-K#$0.+-$0!-%+'82/%#+-./'!/2!($==!(,=-,%$8!2/==/[$0!7)!4=+'-!%$&$'$%+-./'!T?$$;8!*4!('9L!G^^Mo!V+8.=!*4!('9L!G^^Mo!J$(;$%!*4!('5L!G^^Yo!E$@%+!*4!('9L!G^^Yo!>.-+=+!*4!('9L!G^^Yo!?+'!*4!('9L!G^^YU5!B+%-.(=$!7/#7+%0#$'-![+8!2.%8-!,8$0!-/!-%+'82/%#!-@$!.#4/%-+'-!`,$$'8=+'0!(%/4!8,&+%!(+'$!TJ/[$%!+'0!J.%(@L!G^^Ho!J.%(@!+'0!X+%$-Q;.L!G^^MU!7,-!L6$#W()4*$3.8!@+8!+=8/!8.'($!7$$'!,8$0!2/%[email protected]!4,%4/8$5!

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!2.2 Plant selectable markers

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!2.3 Green Florescent Protein (GFP): an excellent reporter for many applications

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A!#,-+'-!<9B!4%/-$.'L!<9B1]ONL!@+8!+!8.'&=$!7+8$!4+.%!8,78-.-,-./'!/2!8$%.'$K]O!-/!-@%$/'.'$!Tc$.#!*4!('5L!G^^[email protected]!#,-+'-!4%/-$.'[email protected]!#+D.#,#!$D(.-+-./'!+-!Y^W!'#!+'0!#+D.#,#!$#.88./'!+-!OGG!'#5!?@$'!$D(.-$0![.-@!7=,$!=.&@-L!U9!)#'3K$D4%$88$0!<9B1]ON!.8!8.D!-.#$8!7%.&@-$%!-@+'![.=0K-)4$!<9BL!+'0!#+##+=.+'!($==K$D4%$88$0!<9B1]ON!.8!,4!-/!GS!-.#$8!7%.&@-$%!-@+'![.=0K-)4$!<9B5!N@$!<9B!-@+-!)/,![.==![/%;![.-@!.'[email protected]!4%+(-.(+=!.8!+!8)'-@$-.(!<9B1]ON!*$%8./'!-@+-!@+8!-@$!2+*/%$0!(/0/'8!/2!@.&@=)!$D4%$88$0!4=+'-!4%/-$.'8!TI,-=$%!*4!('[email protected]!8)'-@$-.(!<9B1]ONL![@.(@!.8!=$88!-@+'!PWj!.0$'-.(+=!-/![.=0K-)4$!<9B!+-!-@$!',(=$/-.0$!=$*$=!7,-!^Sj!.0$'-.(+=!+-!-@$!+#.'/!+(.0!=$*$=L!0.84=+)8!@.&@!=$*$=!$D4%$88./'!.'!U9!)#'3L!4=+'-!+'0!#+##+=.+'!($==8!TI@,.!$-!+=5L!G^^]o!I,-=$%!$-!+=5L!HWWWU5!

!2.4 Transgene expression and gene silencing

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2.6 Description of Practical !C'[email protected]!4%+(-.(+=L![$![.==!-%+'82/%#!4=+'-8!+'0d/%!4=+'-!-.88,$8![.-@!<9BL!+'0!#/'.-/%!

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2.7 DAY 1 – Friday 2 September 2011 !

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I$==!(,=-,%$!-%+'82$(-./'8![.-@!4=+8#.0!:EA!#+)!7$!4$%2/%#$0!2/%!#,=-.4=$!%$+8/'85!9/%!$D+#4=$L!.-!#+)!7$!/2!.'-$%$8-!-/!8-,0)!-@$!$22$(-!/2!/*$%$D4%$88.'&!+!4+%-.(,=+%!&$'$!/'!+!#/=$(,=+%!/%!($==,=+%!4%/($885!A=-$%'+-.*$=)L!.-!#+)!7$!/2!.'-$%$8-!-/!0$-$%#.'$!-@$!8,7($==,=+%!=/(+=.Q+-./'!/2!+!4+%-.(,=+%!&$'$!4%/0,([email protected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

>$4/%-$%!4%/-$.'8!(+'!+=8/!7$!,8$0!-/!+'+=)8$!&$'$!%$&,=+-./'L!7)!(/,4=.'&!-@$!4%/#/-$%!%$&./'!/2!+!&$'$!/2!.'-$%$8-!-/!-@$!&$'$!2/%!+!%$4/%-$%!4%/-$.'5!C2!-@$!%$4/%-$%!.8!$D4%$88$0L!-@$!4%/#/-$%!.8!+(-.*$!.'!-@$!-.88,$!7$.'&!8-,0.$05!N@$!&$'$!2/%!=,(.2$%+8$!.8!(/##/'=)!,8$0!2/%!4%/#/-$%!+88+)85!6,(.2$%+8$!.8!+'!$'Q)#$![@.(@!(/'*$%-8!+!(/=/,%=$88!8,78-%+-$!.'-/!=,(.2$%.'L!+!=,#.'/,8!(/#4/,'0![@.(@!(+'!7$!#$+8,%$0!3,+'-.-+-.*$=)5!

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3.1 Summary of Activities !

G'B'B !78$B$`$A.1&478$D^$E1;31:>16$!GW+#!!!!! ! C'-%/0,(-./'!+'0!+==/(+-./'!/2!&%/,48! !GW5MW+#! B%+(-.(+=!! ! ! ! ! ! ! !K!O4#! ! B%$4+%+-./'!2/%!-%+'82$(-./'85!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!GU!4=+8#.0!:EA!0.&$8-./'L!!! ! ! HU!4+88+&$!+'0!(/,'-!($==8L!4,-!($==8!/'-/!(/*$%8=.485!!

G'B'D !78$D$`$]14+1&478$DB$E1;31:>16$!GW+#! ! :.8(,88./'!/2!=+7/%+-/%)![/%;L![/%;!/'!&%/,4!4%$8$'-+-./'8!GW+#K!O4#! B%+(-.(+=!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!GU!3,+'-.-+-./'!/2!4=+8#.0!:EA!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!HU!-%+'82$(-./'8!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!MU!(/#4,-$%!-+8;!! !! ! !

G'B'G !78$G$`$Z6*478$DG$E1;31:>16$!GW+#! ! :.8(,88./'!/2!=+7/%+-/%)![/%;! !!GW+#KM4#! B%+(-.(+=! ! ! ! ! !! ! B%$4+%$!($==8!2/%!2=,/%$8($'-!#.(%/8(/4)!!!(! ! !! ! ! ! !!

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#$%&'(()"*+,-./0+"12*"30/4.56578"9())" YY"

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Practical schedule !#5A'(!!!! :EA!0.&$8-!+'0!*.8,+=.Q.'&!(,=-,%$0!+0@$%$'-!#+##+=.+'!($==8!Tc$6+U5!N,$8!HW!1$4-! B%$4+%.'&!:EA!2/%!-%+'82$(-./'!!!!!!!!!!!!!!!!!!!!!!!!!!GU!>$8-%.(-./'!$'Q)#$!0.&$8-./'!/2!4=+8#.0!:EA!/*$%'.&@-5!!!!!!!!!!!!!!!!!!!!!!!!!HU!N%)48.'.Q.'&!+0@$%$'-!($==8!! ! !!!!I/,'-.'&!($==8!,8.'&!+!@+$#/()-/#$-$%!! ! !!!!B=+-.'&!($==8!/,-!2/%!-%+'82$(-./'!!! ! !!#5A'_! ! `,+'-.2.(+-./'!/2!4=+8#.0!:EA!+'0!4$%2/%#!-%+'82$(-./'5!?$08!HG!1$4-!! I@$(;.'&!:EA!(/'($'-%+-./'!7)!+&+%/8$!&$=!$=$(-%/4@/%$8.8!!! ! N%+'82$(-./'8!!!! ! c$6+!($==8!<%/,4!ããã555!4"<9BK(+0@$%.'! 6.4/2$(-+#.'$!<%/,4!ããã555!4"<9BK(+0@$%.'! I+B!<%/,4!ããã555!4"<9BK-,7,=.'!! 6.4/2$(-+#.'$!<%/,4!ããã555!4"<9BK-,7,=.'!! I+B!<%/,4!ããã55! 4"<9BKJHM! ! 6.4/2$(-+#.'$!<%/,4!ããã55! 4"<9BKJHM! ! I+B!! ! !! ! !!("==(O0BEFG(G@BE=C(9=GB(<K:=EC/(9(KB(ZT"(:BK;0B=!T[@)RU!(#5A'S!!!! V.8,+=.Q$!-%+'82$(-./'8!,8.'&!2=,/%$8($'($!#.(%/8(/4)!9%.!HM!1$4-! :.8(,88!%$8,=-8!!! ! I/#4,-$%!-+8;!!! ! J+(;&%/,'0!%$+0.'&!!(

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YZ" #$%&'(()"*+,-./0+"12*"30/4.56578"9())"

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3.2 Protocols F9 P$/&13,3X3,6!(2Q*$*,4!)*''1!D9 ?#.,43,6!)*''1!]9 A'(43,6!)*''1!Y9 P$(,1T*)43,6!)*''1!K!?(A!8*4Q#2!f9 P$(,1T*)43,6!)*''1!K!'3&#1#8('!8*4Q#2!^9 e31.('3X3,6!)*''!T'.#$*1)*,)*!

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G'D'B K7316*70&$61S.*614$B=+8#.0!:EA\!á!G!K!O!�&!4$%!-%+'82$(-./'!>$8-%.(-./'!$'Q)#$\!U)#>CL!>"!7,22$%!1-$%.=$!?+-$%!A&+%/8$!&$=!$=$(-%/4@/%$8.8!%$3,.%$#$'-8!T+&+%/8$L!NJ"L!1_J>1+2$L!&$=!-%+)L!-+';L!&$=!

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G'D'D JS.*;:1+3$61S.*614$N.88,$!(,=-,%$!@//0!N.88,$!(,=-,%$!.'(,7+-/%!TMP{IL!OKGWj!IZHU!MP{I![+-$%7+-@!GWW{I![+-$%7+-@!C'*$%-$0!#.(%/8(/4$!J$'(@-/4!($'-%.2,&$!c+$#/()-/#$-$%8!+'0!83,+%$!(/*$%8=.48!B.4$-#+'!TBGWWWL!BHWW!+'0!BHWU!9=,/%$8($'-!#.(%/8(/4$!

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3.3 Day 1. Tuesday 20 September 2011

G'G'B !*-1&3*+-$7+4$O.7+3*H8*+-$!"#$1$-!,4!+!0.&$8-!/2!4=+8#.0!:EA!+8!.'!-@$!-+7=$5!!!!!

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G'G'D A68;&*+*C*+-$Ta&;0*33*+-bU$745161+3$/100&$G5 X+;$!,4!+==!#$0.+!+'0!8/=,-./'8!7$2/%$!%$#/*.'&!($==8!2%/#!-@$!.'(,7+-/%!H5 I@$(;!($==8!,'0$%!-@$!#.(%/8(/4$5!:$8(%.7$![@+-!)/,!8$$!.'!)/,%!'/-$7//;5!A%$!+==!($==8!

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3.4 Day 2. Wednesday 21 September 2011

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3.5 Day 3. Friday 23 September 2011 !

G'L'B \*&.70*C*+-$/100$H0.261&/1+/1$!

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3.6 Appendices !!

G5 B=+8#.0!#+48!!

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BIOC7001 – Advanced DNA Techniques 2011_FINAL

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