Supplementary Cloning Lab

8/17/2019 Supplementary Cloning Lab

1/21

!

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

#$%&'&( ') *+, -.%/,)) %0 ')%$1*'&( 1 ),(2,&* %0 345 67)71$$8 1 (,&,9 0.%2 1& %.(1&')2 1&: 21;'&( 27$*'-$, , 345 /1& =, ')%$1*,: =8 .,)*.'/*'%& ,&?82,

:'(,)*'%& %0 (,&%2'/ 345 %. 12-$'0',: 7)'&( @#A> B+, ),(2,&* %0 345 ') *+,& '&),.*,: '&*% '& 1 )21$$ /'./7$1.

345C ;&%D& 1) 1 E,/*%. %. -$1)2':> B+, &,D /%2='&1*'%& %0 345 ') ;&%D& 1) .,/%2='&1&* 345> #$%&'&( 1$$%D)

*+, -.%:7/*'%& %0 $1.(, 12%7&*) %0 -7., 345C D+'/+ /1& *+,& =, 7),: '& 1--$'/1*'%&) )7/+ 1) ),F7,&/'&(C /.,1*'&(

*.1&)(,&'/ 6(,&,*'/1$$8 2%:'0',:9 -$1&*) 1&: 1&'21$)C 1&: *+, -.%:7/*'%& %0 -7.'0',: -.%*,'&) %& 1 $1.(, )/1$,> B+,

*,/+&'F7,) *% /.,1*, 1&: /$%&, .,/%2='&1&* 345C 1&: *+, 1--$'/1*'%&) D+,., .,/%2='&1&* 345 ') 7),: 1.,

/%$$,/*'E,$8 ;&%D& 1) (,&,*'/ ,&('&,,.'&(> B+') $1= D'$$ :,2%&)*.1*, *+, -.%/,)) %0 '&),.*'&( 1 0.1(2,&* %0 345

-.%:7/,: =8 @#A '&*% 1 /$%&'&( E,/*%. 1&: *.1&)0%.2'&( *+1* E,/*%. '&*% =1/*,.'1>

012(&(/ 34&(/ 506

@#A ') 1 F7'/; 1&: -%D,.07$ *,/+&'F7, *% 12-$'08 1 0.1(2,&* %0 345 '& 1 )+%.* 12%7&* %0 *'2,> G'*+'& H7)* 1 0,D

+%7.)C *.'$$'%&) %0 /%-',) %0 1 0.1(2,&* %0 345 /1& =, -.%:7/,:> I%D,E,.C @#A +1) )%2, $'2'*1*'%&)> J'.)*C @#A

.,F7'.,) ),F7,&/,K)-,/'0'/ -.'2,.) '& %.:,. *% (7':, 345 -%$82,.1), *% *+, ),(2,&* %0 345 *% =, 12-$'0',:>

#.,1*'&( *+,), -.'2,.) *+,.,0%., .,F7'.,) *+1* ),F7,&/, '&0%.21*'%& 1=%7* *+, (,&, %0 '&*,.,)* ') ;&%D&> L,/%&:C*+, 01/* *+1* @#A ') 1 E,.8 ),&)'*'E, *,/+&%$%(8 1$)% -.,),&*) 1 $'2'*1*'%&> B+, .'(+* /%&:'*'%&) +1E, *% =, 1/+',E,:

'& %.:,. 0%. *+, -.'2,.) *% 1&&,1$ *% *+, /%..,/* ),(2,&* %0 345 *% 1E%': %00K*1.(,* 12-$'0'/1*'%&>

7$4&/(&(/ #%&'$%4 82% 506

@.'2,.) 1., %&, %0 *+, 2%)* '2-%.*1&* -1.*) %0 1&8 @#A .,1/*'%&C )'&/, *+,8 :,*,.2'&, D+,*+,. *+, -.%-,.

),(2,&* %0 345 D'$$ =, 12-$'0',: %. &%*> @.'2,.) 1., )+%.* 6!MKNO &7/$,%*':, $%&(9C )'&($,K)*.1&:,: -',/,) %0 345C

1$)% ;&%D& 1) %$'(%&7/$,%*':,)C D+'/+ 1&&,1$ 1* ,1/+ ,&: %0 *+, *1.(,* ),F7,&/, *% =, 12-$'0',:> #7..,&*$8C

/%2-1&',) )-,/'1$'?, '& *+, -.%:7/*'%& %0 -.'2,.) 0%. 7), '& @#AC =7* '* ') *+, .,)-%&)'='$'*8 %0 *+, .,),1./+,. *%

:,*,.2'&, *+, ),F7,&/, *+1* *+, /%2-1&8 )+%7$: -.%:7/,> J%. *.1:'*'%&1$ @#AC *D% -.'2,.) 1., :,)'(&,:C ,1/+

%&, 0$1&;'&( *+, *1.(,* ),F7,&/, *% =, 12-$'0',: 6J'(7., PK!9>

9&/3%$ +:;, @.'2,.) 1., :,)'(&,: *% =, /%2-$,2,&*1.8 *% *+, ),F7,&/, 0$1&;'&( ,'*+,. ,&: %0 1 (,&, %0 '&*,.,)*>


2/21

Q

R& *.1:'*'%&1$ @#AC ),F7,&/, '&0%.21*'%& %0 *+, *1.(,* (,&, ') 7),: *% :,)'(& -.'2,.) D+'/+ 1., -,.0,/*$8

/%2-$,2,&*1.8 *% *+, *1.(,* ),F7,&/, 6J'(7., PK!9> I%D,E,.C D+,& ),F7,&/, '&0%.21*'%& %0 *+, *1.(,* (,&, ')

&%* ;&%D&C *+, 1--.%1/+ *% :,)'(&'&( -.'2,.) /1& =, 2%:'0',:>

5& 1$*,.&1*'E, 1--.%1/+ ') -%))'=$, '0

*+, -.%*,'& ),F7,&/, %0 *+, (,&, %0 '&*,.,)* ') ;&%D&> L*1.*'&( D'*+ *+, -.%*,'& ),F7,&/,C '* ') -%))'=$, *% D%.;

=1/; *% *+, -%))'=$, 345 ),F7,&/, 7)'&( *+, (,&,*'/ /%:,> S&, :.1D=1/; *% *+') 1--.%1/+ ') *+1* =,/17), %0 *+,

:,(,&,.1*, &1*7., %0 *+, (,&,*'/ /%:,C *+,., ') 2%., *+1& %&, -%))'=$, 345 ),F7,&/, *+1* D%7$: .,)7$* '& *+,

;&%D& 12'&% 1/': ),F7,&/,>

B+, (,&, *+1* D'$$ =, )*7:',: '& *+') $1= ,&/%:,) 0%. *+, ,&?82, ($8/,.1$:,+8:,KNK-+%)-+1*, :,+8:.%(,&1),

6U5@3I9> U5@3I ') 1 /.'*'/1$ ,&?82, '&E%$E,: '& =%*+ ($8/%$8)') 6J'(7., PKQ9 1&: '& *+, #1$E'& /8/$, 6:1.; .,1/*'%&)*+1* -.%:7/, /1.=%+8:.1*,)9 '& -$1&*)> 37, *% '*) E'*1$ .%$, '& *+,), =1)'/ -.%/,)),)C *+, U5@3I -.%*,'& ') +'(+$8

/%&),.E,: =,*D,,& %.(1&')2)C -1.*'/7$1.$8 '& *+, 1/*'E, )'*, :%21'&) %0 *+, -.%*,'&> B+') /%&),.E1*'%& =,*D,,&

)-,/',) 1$$%D) 7) *% /.,1*, 1 /%$$,/*'%& %0 -.'2,.) *+1* )+%7$: 1&&,1$ D'*+ )%2, )-,/'0'/'*8 *% *+, U5@3I (,&, '&

1&8 )-,/',) %0 -$1&*>

9&/3%$ +:C, B+, -.%/,)) %0 ($8/%$8)')> U5@3I /1*1$8),) *+, /%&E,.)'%& %0 ($8/,.1$:,+8:,KNK-+%)-+1*, '&*% !CN –

=')-+%)-+%($8/,.1*,>


3/21

N

0%$D)&(/ #%&'$%4 82% 506 28 7>< 28 3(?(2@( 4$A3$(.$

B% /$%&, *+, U5@3I (,&, 0.%2 1 E1.',*8 %0 )-,/',) %0 -$1&*)C -.'2,.) &,,: *% =, :,)'(&,: *+1* 1., /%2-$'2,&*1.8

*% /%&),.E,: .,('%&) %0 *+, (,&,> L'&/, ,E,& /%&),.E,: .,('%&) %0 *+, (,&, D'$$ &%* =, ':,&*'/1$ =,*D,,& )-,/',)C

1 =,)* (7,)) %0 *+, (,&, ),F7,&/, ') 21:, =8 /%2-1.'&( *+, 1$'(&2,&* %0 U5@3I (,&,) 0.%2 21&8 .,$1*,:

%.(1&')2) 60'(7., PKN9> 5$*+%7(+ &%* 1$$ *+, =1),) 1., /%&),.E,:C *+, :'00,.,&/,) 1* ,1/+ =1), -%)'*'%& =,*D,,&

)-,/',) ') &%* .1&:%2> B+, 0%7.*+ =1), %0 ,1/+ ),F7,&/, '& J'(7., PKN ') ,'*+,. 1 B %. 1& 5> B+, =1), *+1* ') 2%.,

/%22%&$8 0%7&: 1* ,1/+ -%)'*'%& /1& =, 7),: 0%. :,)'(&'&( *+, -.'2,.C '& *+') /1),C 1 B D%7$: =, 7),:> V)'&( *+')

'&0%.21*'%&C 1 ),* %0 -.'2,.) D'$$ =, :,)'(&,:C ,1/+ /%&*1'&'&( *+, /%&),.E,: =1),) 1* -%)'*'%&) D+,., *+,., ')

$'**$, *% &% E1.'1='$'*8> B+, -%)'*'%&) *+1* 1., E1.'1=$,C )7/+ 1) -%)'*'%& N 6:,&%*,: 1) W9 D'$$ +1E, 1 :'00,.,&* =1),

1* *+1* -%)'*'%& '& ,1/+ -.'2,. '& *+, ),*C '& *+') /1), 1 UC B %. #> B+') 2' B+, U5@3I

(,&, ') :7-$'/1*,: '& )%2, %.(1&')2)C 1&: *+,.,0%., +1) 2%., *+1& %&, ),F7,&/,C ,1/+ D'*+ 1 )$'(+*$8 :'00,.,&*

&12, 1&: 1/.%&82>

5&%*+,. 2,*+%: 0%. /.,1*'&( :,(,&,.1*, -.'2,.) ') *% 7), 12'&% 1/': ),F7,&/, '&0%.21*'%& 0%. 1 -.%*,'&> B+,

:,(,&,.1/8 %0 *+, (,&,*'/ /%:, 60'(7., PKX9 2,1&) *+1* 0%. %&, 12'&% 1/': ),F7,&/,C *+,., 1., 27$*'-$, -%))'=$,

345 ),F7,&/,) *+1* /%7$: -.%:7/, *+, )12, 12'&% 1/': ),F7,&/,> L%C '& %.:,. *% .,:7/, *+, &72=,. %0

:,(,&,.1*, -.'2,.) &,,:,: 0%. @#AC 1 .,('%& %0 *+, -.%*,'& D'*+ 12'&% 1/':) *+1* 1., ,&/%:,: =8 %&$8 ! %. Q

/%:%&) ,1/+ /1& =, ),$,/*,:>


4/21

X

9&/3%$ +:F, B+, (,&,*'/ /%:, K 12'&% 1/':) 1&: *+, 345 /%:%&) 0%. ,1/+>

>$4)$G 506

W8 /.,1*'&( :,(,&,.1*, -.'2,.) *+1* D'$$ &%* &,/,))1.'$8 +1E, ,,> %00K*1.(,*

12-$'0'/1*'%&9> R& %.:,. *% 1::.,)) *+') '))7, 1&: '&/.,1), *+, /+1&/, *+1* D, 1., 12-$'08'&( *+, U5@3I (,&,C D,

D'$$ -,.0%.2 &,)*,: @#A 60'(7., PKY9> R& &,)*,: @#AC *D% .%7&:) %0 @#A 1., -,.0%.2,: ),F7,&*'1$$8> B+, 0'.)* .%7&:

7),) :,(,&,.1*, -.'2,.)> B+') @#A .,1/*'%& ') *+,& *.,1*,: D'*+ ,

B+, ),/%&: @#A .,1/*'%& 7),) *+, 0'.)* @#A -.%:7/*) 1) 1 *,2-$1*, D'*+ 1 &,D ),* %0 -.'2,.) D+'/+ 1&&,1$ *%

.,('%&) '&)':, %0 *+, *1.(,* .,('%& %0 *+, 0'.)* @#A .,1/*'%&> B+, -.'21.8 1:E1&*1(, %0 &,)*,: @#A ') *+1* '0 *+, 0'.)*

),* %0 -.'2,.) 12-$'0',: 345 %7*)':, %0 *+, *1.(,* 345C '* ') 7&$';,$8 *+1* *+, ),/%&: ),* %0 -.'2,.) D'$$ :% *+,

)12,> B+, 2%)* -.%2'&,&* 0.1(2,&* %0 345 12-$'0',: )+%7$: =, %7. (,&, %0 '&*,.,)*>

G, D'$$ 7), *+, -$1&* (,&%2'/ 345 8%7 ')%$1*,: 0.%2 E1.'%7) 0.7'*) 1&: E,(,*1=$,) :7.'&( *+, UZS $1= *% 1**,2-*

*% /$%&, *+, U5@3I (,&, 0.%2 ,1/+ )12-$,> 5) 1 /%&*.%$ 0%. *+, &,)*,: @#A 0%. U5@3IC D, D'$$ =, 7)'&( (,&%2'/

345 0.%2 !"#$%&'()%) +,#-%#.#C 1 2,2=,. %0 *+, 27)*1.: 012'$8 1&: *+, 2%)* ,

!"#$%&'()%) ') 1& ':,1$ 2%:,$ -$1&* :7, *% '*) )21$$ (,&%2, D+'/+ +1) =,,& 07$$8 ),F7,&/,: 6!QY Z=9 1&: '*) .1-':

$'0, /8/$, %0 %&$8 [ D,,;)> !"#$%&'()%) /%&*1'&) 1 012'$8 %0 X :'00,.,&* U5@3I (,&,) 60'(7., PKN9> B+, -.'2,.

2'


5/21

Y

9&/3%$ +:H, 4,)*,: @#A -.%/,))


6/21

[

9&/3%$ +:I, T B+, 1:E1&*1(, %0 '&),.*'&(

%7. 345 '&*% 1 -$1)2': ') *+1* *+, =1/*,.'72 /1& :% *+, D%.; %0 .,-$'/1*'&( *+, 345> A,-$'/1*'%& %0 -$1)2':)

%//7.) '&:,-,&:,&* %0 *+, *'2'&( %0 *+, .,-$'/1*'%& %0 *+, =1/*,.'1$ /+.%2%)%2,> 3,-,&:'&( %& *+, *8-, %0

-$1)2':C 1 )'&($, =1/*,.'72 /1& -.%:7/, +7&:.,:) %0 /%-',) 67- *% POO_9 %0 *+, )12, -$1)2':> W8 (.%D'&( $1.(,

&72=,.) %0 *+, =1/*,.'1 '& /7$*7.,C 21&8 2%., /%-',) %0 *+, 345 1., -.%:7/,: *+1& *+.%7(+ *+, -.%/,)) %0 @#A>

1

2

3

4

6

7

5

8

9

1

2

3

4

6

7

5

8

9


7/21

P

B+') $1.(, 12%7&* %0 345 /1& =, -7.'0',: *% 1&1$8?, 07.*+,. 6)7/+ 1) 0%. ),F7,&/'&( *+, (,&,9 %. *% 21&'-7$1*,

07.*+,. 6)7/+ 1) :7.'&( *+, -.%:7/*'%& %0 UZS /.%-)9>

B+, -$1)2': D, D'$$ '&),.* %7. @#A -.%:7/* '&*% ') *+, -`,*!>Q =$7&*,: E,/*%. 60'(7., PKP9> B+, E,/*%. +1) ),E,.1$

0,1*7.,) *+1* 21;, '* ':,1$ 0%. /$%&'&(> J'.)*C '* ') 1 =$7&*,: E,/*%.C D+'/+ 2,1&) D, /1& '&),.* %7. @#A -.%:7/*)

'&*% *+, E,/*%. D'*+ 2'&'21$ -.,-1.1*'%& %0 *+, @#A -.%:7/*> L,/%&:C *+, -$1)2': /%&*1'&) *+,/0'1234 (,&,> B+')

(,&, /%:,) 0%. *+, T/%XPR .,)*.'/*'%& ,&?82,C D+'/+ D+,& ,

9&/3%$ +:+, -`,*!>Q =$7&*,: E,/*%.

G, D'$$ 7), *+, -.%/,)) %0 $'(1*'%& *% '&),.* %7. @#A -.%:7/* '&*% *+, -`,*!>Q -$1)2':> ]'(1*'%& ') *+, -.%/,)) %0

H%'&'&( *D% -',/,) %0 $'&,1. 345 '&*% 1 )'&($, -',/,> 345 $'(1), 6*+, ,&?82, *+1* /1*1$8?,) *+, $1)* )*,- '& 345

.,-$'/1*'%&9 ') 7),: *% H%'& *+, *D% -',/,) %0 345 *%(,*+,.> B+, 2%)* /%22%&$8 7),: $'(1), /%2,) 0.%2 *+,

=1/*,.'%-+1(, BX 1&: ') &12,: BX 345 $'(1),> 5) D'*+ 1$$ 345 $'(1),)C *+') ,&?82, /1*1$8?,) *+, 0%.21*'%& %0 1

-+%)-+%:',)*,. =%&: =,*D,,& *+, N’KSI %0 %&, -',/, *% 345 1&: *+, Y’K-+%)-+1*, %& *+, ),/%&: -',/, %0 345>

R22,:'1*,$8 0%$$%D'&( $'(1*'%&C *+, $'(1*,: -$1)2': ') *+,& *.1&)0%.2,: '&*% =1/*,.'1>


8/21

M

!"$%.&4$ +, -$($)&. $(/&($$%&(/

]T5A4R4U SW`T#BRaTL

3,)/.'=, *+, =1)'/ )*,-) %0 /$%&'&(

#%2-1., 1&: /%&*.1)* )'2-$, 1&: &,)*,: @#A

#%2-1., *+, *.1&)0%.21*'%& ,00'/',&/8 %0 1& '&*1/* -$1)2': *% 1 $'(1*,: -$1)2':>

@AS#T3VATL

@$1&* (,&%2'/ DNA for this lab will come from the “test food” DNA isolated in section 5.1 of Experiment 5:

U,&,*'/1$$8 2%:'0',: %.(1&')2)> J%. *+, @CR reactions listed here, the “test food” must be a 0.,)+ E,(,*1=$, %.

-$1&* %0 )%2, ;'&:b -.%/,)),: 0%%:) D'$$ &%* +1E, 1:,F71*, 345 *% ,&)7., -.%-,. 12-$'0'/1*'%& %0 *+, *1.(,*

(,&,C 7!89:>

+O; P$))&(/ 3# &(&)&D1 506 %$D.)&2(4

B+, )*7:,&*) 1* ,1/+ *1=$, D'$$ ),* 7- @#A .,1/*'%&) 1) 1 (.%7-> S& 8%7. '/,C 8%7 )+%7$: +1E, *+.,, *7=,) 0%. *+,

/%&*.%$ 345 *,2-$1*,)^ %&, ') *+, &,(1*'E, /%&*.%$ 1&: /%&*1'&) )*,.'$, D1*,.C *+, ),/%&: *7=, /%&*1'&)

5.1=':%-)') (,&%2'/ 345C 1&: *+, *+'.: ') 1 -%)'*'E, /%&*.%$ /%&*1'&'&( *+, -U5@ -$1)2': 345> 5) 1 (.%7-C 8%7

D'$$ 1$)% ),* 7- %&, ,

]1=,$ *+, X @#A *7=,) 1* 8%7. *1=$, D'*+ *+, &72=,.) !KX 1&: '&'*'1$ *+,2 %& *+, *%- 1&: )':, )% *+1* *+,8

/1& =, ':,&*'0',: &, A,2,2=,.C $1=,$) %& *+, *%- %0 *+, *7=, 1., $';,$8 *% =, $%)* :7, *% *+,

+,1*,: $': %0 *+, @#A 21/+'&,> c,,- *+, *7=,) %& '/, 0%. *+, &,

N>

J$'/; *% *+%.%7(+$8 2'< *+, /%&*,&*) %0 *+, Q< =$7, '1)*,.''< D'*+ &&'*'1$ #.'2,.) 6Q< ZZR@9 *7=, 1&:

/,&*.'07(, !O ),/ *% 0%./, /%&*,&*) *% *+, =%**%2 %0 *+, *7=,>

X>

@'-,* QO $ %0 *+, ZZR@ '&*% ,1/+ @#A *7=, %& '/,>

0D3)&2(, W, )7., &%* *% :')*7.= *+, R&)*1U,&, -,$$,* D+'$, *.1nsferring the “test food” DNA sample to the

@#A .,1/*'%&>

Y>

V)'&( 1 0.,)+ *'- 0%. ,1/+ )12-$,C 1:: QO $ %0 *+, 1--.%-.'1*, 345 *,2-$1*, *% ,1/+ *7=, 7)'&( *1=$, PK

! 1) 1 (7':,> 7/.+- A,/1- *7=,)>


9/21

\

JDB1$ +:;O R&'*'1$ @#A .,1/*'%& /%2-%&,&*) 6=$7,9

B7=,

$1=,$L12-$, &12,

W$7, Z1)*,.

Z'<345 B,2-$1*,

B%*1$

a%$72,

!4,(1*'E, /%&*.%$

6)*,.'$, D1*,.9QO $ QO $ D1*,. XO $

Q !"#$%&'()%) (345

N@%)'*'E, /%&*.%$ -U5@

-$1)2':

XPlant (“test food”)

(345

[>

],1E, 8%7. )12-$,) %& '/, 7&*'$ 8%7. B5 1);) 8%7 *% =.'&( 8%7. )12-$,) *% *+, *+,.21$ /8/$,. 0%. $%1:'&(>

J%. %.(1&'?1*'%&1$ -7.-%),)C 8%7. B5 D'$$ -$1/, 8%7. *7=,) '& &72,.'/1$ %.:,. 1&: '& )*.1'(+* .%D) '&)':,

*+, *+,.21$ /8/$,. 1//%.:'&( *% *+, /+1.* -.%E':,:> L'(& *+, )+,,* &, J'&')+,: )12-$,) D'$$ =, )*%.,: '&

*+, 0.,,?,. 7&*'$ *+, &,

M>

#$,1& 8%7. )*1*'%& =8 :')-%)'&( %0 '/, '& *+, )'&;>

+O; P$.)&2( %$N&$@

!> G+1* ') *+, -7.-%), %0 7)'&( :,(,&,.1*, -.'2,.) 0%. *+') '&'*'1$ @#A .,1/*'%&d

Q> G+8 ') *+, U5@3I (,&, 1 (%%: /1&:':1*, (,&, *% 1**,2-* *% 12-$'08 0.%2 1 E1.',*8 %0 :'00,.,&* -$1&*)d

+OC P$))&(/ 3# ($4)$G 506 %$D.)&2(4

5(1'&C 1) 1 (.%7-C 8%7 D'$$ -.,-1., X @#A )12-$,)> S&, &,)*,: @#A D'$$ =, -,.0%.2,: 0%. ,1/+ (345 )12-$,

12-$'0',: '& *+, '&'*'1$ .%7&: %0 @#A 6'&'*'1$ @#A )12-$,) Q 1&: X %&$89> R& 1::'*'%&C 8%7 D'$$ -,.0%.2 %&, -%)'*'E,

/%&*.%$ 7)'&( ($@ /%&*.%$ -U5@ -$1)2': 345 1&: %&, &,(1*'E, /%&*.%$ D'*+ )*,.'$, D1*,. '&)*,1: %0 345 *,2-$1*,>

!>

A,*.',E, '&'*'1$ @#A )12-$,) C D(G F *+1* 8%7 -.,-1.,: -.,E'%7)$8> ],1E, )12-$,) ! 1&: N '& *+, 0.,,?,. *%

=, 1&1$8?,: %& 1 (,$ &, V), B1=$, PK! *% :,*,.2'&, *+, /%&*,&*) %0 ,1/+ @#A *7=,> L12-$,) Q 1&:

X )+%7$: =, *+, !"#$%&'()%) (345 1&: *,)* 0%%: @#A )12-$,)C .,)-,/*'E,$8> @$1/, *7=,) Q 1&: X %& '/,>

Q>

@#A .,1/*'%&) Q 1&: X /%&*1'& (,&%2'/ 345C @#A -.%:7/* 1&: 1&8 7&'&/%.-%.1*,: -.'2,.) 0.%2 *+, '&'*'1$

@#A .,1/*'%&> R& %.:,. 0%. *+, &,)*,: @#A -.'2,.) *% D%.; %-*'21$$8C D, 27)* .,2%E, *+, 7&'&/%.-%.1*,:

'&'*'1$ -.'2,.) 7)'&( , V)'&( 1 0.,)+ *'- ,1/+ *'2,C -'-,* ! 7$ %0 ,


10/21

!O

N>

R&/7=1*, *+, ,

X>

I,1* '&1/*'E1*, *+, ,

P>

V)'&( 1 0.,)+ *'- ,1/+ *'2,C 1:: Q $ %0 ,1/+ , @$1/, *+,), *7=,) D'*+ *+, :'$7*,: 345 *,2-$1*,) %& '/,>

0D3)&2(, A,*7.& *+, *7=,) $1=,$$,: Q 1&: X *% *+, 0.,,?,. D'*+ *7=,) ! 1&: N *% 1&1$8?, %& *+, 1(1.%), (,$ &,

M>

a%.*,< %. 0$'/; *+, *7=,) $1=,$$,: “,

\>

]1=,$ *+, X @#A *7=,) 1* 8%7. *1=$, D'*+ *+, &72=,.) YKM 1&: '&'*'1$ *+,2 %& *+, *%- 1&: )':, )% *+1* *+,8

/1& =, ':,&*'0',: &, c,,- *+, *7=,) %& '/, 0%. *+, &,

!O>

J$'/; *% *+%.%7(+$8 2'< *+, /%&*,&*) %0 *+, Q< 8,$$%D '1)*,.''< D'*+ (,)*,: #.'2,.) 6Q< ZZ4@9 *7=, 1&:

/,&*.'07(, !O ),/ *% 0%./, /%&*,&*) *% *+, =%**%2 %0 *+, *7=,>

!!>

@'-,* QO $ %0 *+, ZZ4@ '&*% ,1/+ @#A *7=, %& '/,>

!Q>

V)'&( 1 0.,)+ *'- 0%. ,1/+ )12-$,C 1:: QO $ %0 *+, 1--.%-.'1*, 345 *,2-$1*, *% ,1/+ *7=, 7)'&( *1=$, PK

Q 1) 1 (7':,> 7/.+- A,/1- *7=,)>

!N>

],1E, 8%7. )12-$,) %& '/, 7&*'$ 8%7. B5 1);) 8%7 *% =.'&( 8%7. )12-$,) *% *+, *+,.21$ /8/$,. 0%. $%1:'&(>

J%. %.(1&'?1*'%&1$ -7.-%),)C 8%7. B5 D'$$ -$1/, 8%7. *7=,) '& &72,.'/1$ %.:,. 1&: '& )*.1'(+* .%D) '&)':,

*+, *+,.21$ /8/$,. 1//%.:'&( *% *+, /+1.* -.%E':,:> L'(& *+, )+,,* &, J'&')+,: )12-$,) D'$$ =, )*%.,: '&

*+, 0.,,?,. 7&*'$ *+, &,

!Y>

#$,1& 8%7. )*1*'%& =8 :')-%)'&( %0 '/, '& *+, )'&;>


11/21

!!

JDB1$ +:CO 4,)*,: @#A .,1/*'%& /%2-%&,&*) 68,$$%D9

B7=,

$1=,$

L12-$, 6*,2-$1*,9

&12,

Q$112@

Z1)*,. Z'<345 B,2-$1*,

B%*1$

a%$72,

Y4,(1*'E, /%&*.%$

6)*,.'$, D1*,.9QO $ QO $ D1*,. XO $

[

T


12/21

!Q

Q>

A,*.',E, *+, @#A )12-$,) 8%7 -.,-1.,: -.,E'%7)$8> V), *1=$, PK! 1&: PKQ *% :,*,.2'&, *+, /%&*,&*) %0

,1/+ )21$$ *7=,> R0 8%7 -.,-1.,: 8%7. @#A) '& *+, /%..,/* %.:,.C *+, &72=,.,: $': '&:'/1*,) *+,

/%2-%&,&*) %0 ,1/+ *7=,>

N>

@$1/, @#A *7=,) '&*% 1:1-*,. *7=,) 1&: -7$),K)-'& 0%. 1=%7* N ),/%&:) *% =.'&( 1$$ *+, $'F7': *% *+, =%**%2>

Z1;, )7., *+, 21/+'&, ') =1$1&/,: 1&: *+, .%*%. $': ') D,$$ ),/7.,:>

X>

c,,- 8%7. )12-$,) %& '/, :7.'&( *+, -.,-1.1*'%& 0%. ,$,/*.%-+%.,)')>

>2)$, L12-$,) !KX 1&: YKM D'$$ =, *.,1*,: :'00,.,&*$8> @18 /1.,07$ 1**,&*'%& :7.'&( *+, &,

Y>

@.,-1., )12-$,) YKM 0'.)*> L'&/, *+, &,)*,: @#A -.%:7/*) 6*7=,) YKM9 D'$$ 1$)% =, 7),: 0%. $'(1*'%& '&*% *+,

-`,*!>Q -$1)2':C D, 27)* 1$'F7%* 1 )21$$ 12%7&* %0 ,1/+ )12-$, '&*% &,D *7=,) *% /%2='&, D'*+ $%1:'&(

:8, 6]39 0%. (,$ ,$,/*.%-+%.,)')> ]1=,$ *+, @#A *7=,) '& 8%7. 2'/.%*7=, .1/; D'*+ *+, 0%$$%D'&( &72=,.)^

5D, 6D, 7D, 8D. The “D” mark w'$$ '&:'/1*, *+,), 1., *+, *7=,) *% 1:: $%1:'&( :8, *%>

[>

5:: !O $ %0 $%1:'&( :8, 6]39 *% ,1/+ %0 *+, *7=,) '& *+, 2'/.%*7=, .1/; 6Y3C [3C P3C M39>

P>

V)'&( 1 0.,)+ *'- 0%. ,1/+ )12-$,C -'-,* !O $ %0 *+, 1--.%-.'1*, @#A )12-$, 6YKM %&$8_9 '&*% *+,

appropriate “D” tube containing loading dye. U,&*$8 -'-,* 7- 1&: :%D& *% 2'< .,1(,&*)>

U'#2%)D()VV, J%. )12-$,) YKMC ;,,- *+, .,21'&:,. %0 *+, @#A .,1/*'%& %& '/, 0%. -7.'0'/1*'%& '& 1 $1*,. )*,->

M>

4%D -.,-1., )12-$,) !KX> V)'&( 1 0.,)+ *'- 0%. ,1/+ 1::'*'%&C 1:: !O $ %0 S.1&(, U $%1:'&( :8, 6]39:'.,/*$8 *% ,1/+ @#A )12-$,> U,&*$8 -'-,* 7- 1&: :%D& *% 2'< .,1(,&*)>

\>

]%1: QO $ %0 )12-$,) !KMC %&, )12-$, -,. $1&,> R* D'$$ =, ,1)',)* *% 21;, /%2-1.')%&) '0 *+, '&'*'1$ 1&:

&,)*,: @#A )12-$,) %0 ,1/+ 345 *,2-$1*, 1., $%1:,: &, T1/+

*,12 2,2=,. )+%7$: $%1: 1* $,1)* %&, )12-$,> B+,., 1., 1 0,D ,


13/21

!N

JDB1$ +:EO U,$ $%1:'&( .,/%.:

]1&, &72=,. L12-$, $%1:,:

! Don’t load anything here!

Q

N

X

Y

[

P

M

\

!O

!!

!Q

!N

!X Don’t load anything here!

!O>

A7& *+, (,$ 1* !YOa 0%. NOKXY 2'&7*,)>

!!>

B7.& %00 *+, -%D,. )7--$8> V&-$7( *+, ,$,/*.%:, $,1:)>

!Q>

A,2%E, *+, /%E,. 0.%2 *+, (,$ 1--1.1*7)>

0D3)&2(, B+') (,$ ') 1 $%D,. -,./,&*1(, %0 1(1.%), *+1& D, +1E, =,,& 7)'&( )% 01. '& *+, $1=)> B+, (,$ D'$$ =,

2%., 0.1('$, *+1& -.,E'%7) (,$)> V), /1., :7.'&( 1&8 +1&:$'&( )*,-)>

!N>

3,)'(&1*, %&, (.%7- 2,2=,. *% +1&:$, *+, (,$> B+1* -,.)%& 27)* 1$D18) D,1. ($%E,) D+'$, +1&:$'&( *+,

(,$C *+, )*1'&'&( *.18 1&: *+, )*1'&'&( )%$7*'%&>

!X>

L*1'& 1&: :,)*1'& *+, (,$ 1) '& T[> A,2,2=,. *+1* 1 $%&(,. :,)*1'& ('E,) ,1)',. *%

:')*'&(7')+ =1&:)> L;,*/+ 8%7. .,)7$*) &%DC =7* (,$) 218 =, $,0* '& D1*,. D'*+ ;/.+-/ 1('*1*'%& *% 1&1$8?,

1(1'& 1* *+, ,&: %0 $1=>

!Y>

L;,*/+ 8%7. .,)7$*) '& B1=$, PKX>


14/21

!X

JDB1$ +:FO R&'*'1$ 1&: &,)*,: @#A .,)7$*)>

+OE P$.)&2( %$N&$@

!> #%2-1., 1&: /%&*.1)* *+, .,)7$*) 8%7 %=*1'&,: 0%. *+, !"#$%&'()%) gDNA template and the “test food”

*,2-$1*,> G,., )'2'$1. -.%:7/*) 6'& *,.2) %0 )'?, 1&: &72=,. %0 =1&:)9 %=*1'&,: 0%. *+,), *D% *,2-$1*,)d

Q> #%2-1., 1&: /%&*.1)* *+, .,)7$*) 8%7 %=*1'&,: 0.%2 *+, '&'*'1$ @#A .,1/*'%&) /%2-1.,: *% *+, &,)*,: @#A

.,1/*'%&)>

+OF 53%&8&.D)&2( 28 ($4)$G 506 #%2G3.)4 K=$112@L

W,0%., -.%/,,:'&( *% $'(1*, %7. @#A -.%:7/*) '&*% *+, -`,*!>Q -$1)2': E,/*%.C )21$$,. '2-7.'*',)C )7/+ 1)

7&'/%.-%.1*,: -.'2,.) %. :4B@) 1&: B1F -%$82,.1), 27)* =, .,2%E,:>

B+') -1.* %0 *+, $1= D'$$ =, /%2-$,*,: '& (.%7-) %0 0%7. 1* ,1/+ *1=$,> T1/+ (.%7- %0 )*7:,&*) D'$$ -7.'08 *+, @#A

products of only the Arabidopsis and “test food” nested PCR reactions (PCR tubes la=,$,: [ 1&: MC ),, *1=$, PKQ>9

5* 8%7. )*1*'%&C 8%7 D'$$ 0'&: 1 )21$$ 2'/.%*7=, .1/; *+1* /%&*1'&) Q ,1/+ %0 @#A c$,,& )-'& /%$72&)C Q>O 2$ D1)+

*7=,)C 1&: !>Y 2$ /%$$,/*'%& *7=,)>

!>

]%/1*, *+, *7=,) $1=,$$,: [ 1&: M 0.%2 8%7. @#A *7=,) %& '/,> V), *1=$, PKQ *% :,*,.2'&, *+, /%&*,&*)

%0 ,1/+ )21$$ *7=,> R0 8%7 -.,-1.,: 8%7. @#A) '& *+, /%..,/* %.:,.C *+, &72=,.,: $': '&:'/1*,) *+,

/%2-%&,&*) %0 ,1/+ *7=,>

Q>

@$1/, @#A *7=,) '&*% 1:1-*,. *7=,) 1&: -7$),K)-'& 0%. 1=%7* N ),/%&:) *% =.'&( 1$$ *+, $'F7': *% *+,

=%**%2> Z1;, )7., *+, 21/+'&, ') =1$1&/,: 1&: *+, .%*%. $': ') D,$$ ),/7.,:>

1

2

3

4

6

7

5

8

9

10

11

12

13

14


15/21

!Y

N>

c,,- 8%7. )12-$,) %& '/, :7.'&( *+, -.,-1.1*'%& 0%. -7.'0'/1*'%&>

X>

]1=,$ *+, Q )-'& /%$72&) 1* 8%7. *1=$, D'*+ *+, &72=,.) [ 1&: M 1&: '&'*'1$ *+,2 !" $%& '()& )% *+1*

*+,8 /1& =, ':,&*'0',:>

Y>

]1=,$ *+, Q>O 2$ D1)+ *7=,) 1* 8%7. *1=$, D'*+ *+, &72=,.) [G 1&: MG 1&: '&'*'1$ *+,2 %& *+, *%- 1&:

)':, )% *+1* *+,8 /1& =, ':,&*'0',:> The “W” indicates these are the wash tubes.

[>

]1=,$ *+, !>Y 2$ /%$$,/*'%& *7=,) 1* 8%7. *1=$, D'*+ *+, &72=,.) [@ 1&: M@ 1&: '&'*'1$ *+,2 %& *+, *%-

1&: )':, )% *+1* *+,8 /1& =, ':,&*'0',: &,

P>

A,)7)-,&: *+, .,)'& '& *+, )-'& /%$72& =8 E%.*,

\>

#,&*.'07(, *+, /%$72&) '& D1)+ *7=,) 0%. ! 2'&7*, '& *+, 2'/.%/,&*.'07(, 1* PNY < ( 6iQMOO .-29> B+,

B5 D'$$ +,$- /%%.:'&1*, *+, $%1:'&( %0 *+, 2'/.%/,&*.'07(, 1* *+') )*,->

!O>

A,*.',E, 8%7. )-'& /%$72&) '& D1)+ *7=,) 1&: .,2%E, *+, D1)+ *7=,> @$1/, *+, )-'& /%$72&) '& *+,

1--.%-.'1*, ;OH'1 .211$.)&2( )3B$4 K1DB$1$G I5 D(G X5LO

!!>

5--$8 *+, 07$$ NO $ %0 ,1/+ @#A .,1/*'%& *% *+, *%- /,&*,. %0 *+, /%$72& =,: '& *+, 1--.%-.'1*, /%$72&>

W, /1.,07$ &%* *% :')*7.= *+, /%$72& =,:> L,, 0'(7., PKM =,$%D>

9&/3%$ +:XO 5--$8'&( @#A )12-$, *% )-'& /%$72&>

!Q>

#,&*.'07(, *+, /%$72&) '& !>Y 2$ /%$$,/*'%& *7=,) 0%. Q 2'&7*,) '& *+, 2'/.%/,&*.'07(, 1* PNY < ( 6iQMOO.-29> B+, B5 D'$$ +,$- /%%.:'&1*, *+, $%1:'&( %0 *+, 2'/.%/,&*.'07(, 1* *+') )*,->

!N>

L1E, *+, -7.'0',: )12-$,) D+'/+ 1., '& *+, =%**%2 %0 *+, !>Y 2$ /%$$,/*'%& *7=,) 6[@ 1&: M@9> B+,),

)12-$,) D'$$ =, 7),: 0%. *+, $'(1*'%& .,1/*'%& '& *+, &,


16/21

![

+OH M&/D)&2( 28 ($4)$G 506 #%2G3.)4 &()2 #Y$);OC N$.)2%

The first reaction for this procedure will remove the 3’ nucleotide overhang from the purified PCR products you

-.,-1.,: -.,E'%7)$8 6[@ 1&: M@9 7)'&( *+, -.%%0.,1:'&( -%$82,.1),> S&/, *+, @#A -.%:7/* ') =$7&*,:C *+, ),/%&:

.,1/*'%& D'$$ $'(1*, *+, @#A -.%:7/*) '&*% *+, -`,*!>Q E,/*%.>

B+') -1.* %0 *+, $1= D'$$ =, /%2-$,*,: '& (.%7-) %0 0%7. 1* ,1/+ *1=$,> S& 8%7. '/,C 8%7 D'$$ 0'&: $'(1*'%& .,1/*'%&=700,. 6]WC /%&*1'&) *+, -.%%0.,1:'&( -%$82,.1),9 1&: 1* 8%7. )*1*'%& %& 1 2'/.%*7=, .1/;C 8%7 D'$$ 0'&: N @#A

*7=,)> B+, BX 345 $'(1), 6BX9 1&: *+, -`,*!>Q =$7&*,: E,/*%. 6a9 1., %& '/, 1&: D'$$ =, )7--$',: =8 *+, *,1/+'&(

1))')*1&* D+,& 8%7 1., .,1:8 *% 7), *+,2>

>2)$, B+,., 1., )*,-) '& *+') -1.* %0 *+, -.%/,:7., D+'/+ .,F7'., -'-,**'&( E,.8 )21$$ 12%7&*) %0 $'F7': 6=,*D,,&

>Y $ 1&: ! $9> A,2,2=,. *+1* *+') ') 1 E,.8 )21$$ 12%7&*C 1&: *+1* '* 218 0,,$ $';, 1 E,.8 )21$$ 2%E,2,&* %0 *+,

-$7&(,. D+,& 8%7 *1;, 7- *+, $'F7':> B1;, 1 $%%; 1* *+, -'-,* *'- *% ,&)7., *+1* 8%7 +1E, -'-,**,: *+, /%..,/*

E%$72, %0 .,1(,&*> I1E, 1 (.%7- 2,2=,. *+1* ') /%&0':,&* D'*+ -'-,**'&( /%2-$,*, *+') -1.* %0 *+, -.%/,:7., *%

/%&),.E, .,1(,&*)>

!>

A,*.',E, *+, -7.'0',: @#A )12-$,) 6[@ 1&: M@9 *+1* 8%7 -.,-1.,: -.,E'%7)$8>

Q>

]1=,$ *+, *+.,, @#A *7=,) [C M 1&: 4# 0%. &,(1*'E, /%&*.%$ 6*+') .,1/*'%& D'$$ /%&*1'& %&$8 E,/*%.9 1&:

'&'*'1$ *+,2 %& *+, *%- 1&: )':, )% *+1* *+,8 /1& =, ':,&*'0',:> c,,- *+, *7=,) %& '/, 0%. *+, &, j%7 D'$$ 1:: $'(1*'%& =700,. 1&: @#A -.%:7/* 0'.)* 1&:

'&/7=1*, 0%. *+, -.%%0.,1:'&( .,1/*'%& 6)*,-) [K\9C *+,& 8%7 D'$$ 1:: *+, E,/*%. 1&: $'(1), 6)*,- !O9>

N>

#,&*.'07(, *+, *7=, /%&*1'&'&( $'(1*'%& .,1/*'%& =700,. 6]W9 0%. !O ),/%&:) *% 0%./, /%&*,&*) *% *+, =%**%2

%0 *+, *7=,>

X>

@'-,* M $ %0 *+, $'(1*'%& .,1/*'%& =700,. 6]WC /%&*1'&) -%$82,.1),9 '&*% ,1/+ @#A *7=, %& '/,>

Y>

V)'&( 1 0.,)+ *'- 0%. ,1/+ )12-$,C 1:: ! $ %0 *+, 1--.%-.'1*, @#A -.%:7/* *% ,1/+ *7=, 7)'&( *1=$, PKY 1)

1 (7':,> JR$ ($/D)&N$ .2()%21 D1%$DG= RD4 ; 1 28 @D)$% DGG$G 82% =23O T1/+ .,1/*'%& &%D /%&*1'&) \ $>

7/.+- A,/1- *7=,)>

JDB1$ +:H> ]'(1*'%& .,1/*'%& /%2-%&,&*)>

B7=,

$1=,$L12-$, &12,

]'(1*'%&

=700,.@#A -.%:7/*

-`,*!>Q

E,/*%.

BX 345

$'(1),

J'&1$

a%$72,

4#4,(1*'E, /%&*.%$

6E,/*%. %&$89M $ ! $ D1*,. >Y $ >Y $ !O $

[ !"#$%&'()%) (345

MPlant (“test food”)

(345

[>

#,&*.'07(, '& *+, 2'/.%/,&*.'07(, 0%. !O ),/%&:) *% 0%./, /%&*,&*) *% *+, =%**%2 %0 *+, *7=,>


17/21

!P

P>

@$1/, *+, *7=, 1* POe# 0%. Y 2'&7*,)>

M>

@$1/, *+, *7=, %& '/, *% /%%$ 0%. Q 2'&7*,)>

\>

#,&*.'07(, '& *+, 2'/.%/,&*.'07(, 0%. !O ),/%&:) *% 0%./, /%&*,&*) *% *+, =%**%2 %0 *+, *7=,> @$1/, *+,

*7=, 1* .%%2 *,2-,.1*7.,>

!O>

V)'&( *+, )12, *7=,) /%&*1'&'&( *+, -.%%0.,1:'&( .,1/*'%&) 8%7 -.,-1.,: '& )*,-) [K\C 1&: 1 0.,)+ *'- 0%.

,1/+ )12-$,C 1:: >Y $ ,1/+ %0 *+, -`,*!>Q E,/*%. 1&: BX 345 $'(1), *% ,1/+ .,1/*'%&> T1/+ .,1/*'%& &%D

/%&*1'&) !O $6 7/.+- A,/1- *7=,)>

!!>

#,&*.'07(, '& *+, 2'/.%/,&*.'07(, 0%. !O ),/%&:) *% 0%./, /%&*,&*) *% *+, =%**%2 %0 *+, *7=,>

!Q>

R&/7=1*, *+, $'(1*'%& .,1/*'%&) 1* .%%2 *,2-,.1*7., 0%. !O 2'&7*,)>

!N>

@$1/, *+, $'(1*'%& .,1/*'%&) %& '/, 7&*'$ .,1:8 *% -.%/,,: D'*+ *+, *.1&)0%.21*'%& )*,- 6),/*'%& P>[9>

+OI J%D(482%'D)&2( 28 1&/D)&2( %$D.)&2(4

>2)$, 7$#$(G&(/ 2( =23% 1DB 4$.)&2(S )R&4 )%D(482%'D)&2( #%2.$G3%$ @&11 B$ #$%82%'$G G&88$%$()1=O JR$ J< @&11

1$) =23 ?(2@ @R&.R #%2.$G3%$ )2 #%2.$$G @&)RO

+OID U8 =23 @&11 B$ #$%82%'&(/ )R$ )%D(482%'D)&2( D4 @$ G&G 82% )R$ #-MZ #1D4'&GS 34$ )R$ #%2.$G3%$ B$12@,

!> ]1=,$ *+, 0%7. -'&; 0'$$,: 2'/.%*7=,) %& '/, 4# 0%. &,(1*'E, /%&*.%$C [C M 1&: @# 0%. -%)'*'E, /%&*.%$ 6-U]S

-$1)2':9 1&: '&'*'1$ *+,2 %& *+, *%- 1&: )':, )% *+1* *+,8 /1& =, ':,&*'0',:> c,,- *+, *7=,) %& '/, 0%. *+, &,

JDB1$ +:ID> B.1&)0%.21*'%& .,1/*'%& /%2-%&,&*)>

B7=,k-$1*,

$1=,$L12-$, &12,

]'(1*'%&

.,1/*'%&

#1$/'72

/+$%.':,

)%$7*'%&

J'&1$

a%$72,

4#4,(1*'E, /%&*.%$

6E,/*%. %&$89Y $ QYO $ QYY $

[ !"#$%&'()%) (345 QYO $


(345

QYO $

@#-U]S

6-%)'*'E, /%&*.%$9! $ QYO $

Q>

]1=,$ *+, 8,$$%D 2'/.%*7=, ]W 6&7*.',&* =.%*+9> R* 1$.,1:8 /%&*1'&) *+, &7*.',&* =.%*+ &,,:,: 0%. *+,

.,/%E,.8 )*1(,> c,,- *+') *7=, %& *+, 2'/.%*7=, .1/;>


18/21

!M

N>

J%$$%D'&( )*,.'$, *,/+&'F7,C 7), 1 &,D '&%/7$1*'%& $%%- *% -'/; 7- QKN )'&($, /%$%&',) %0 =1/*,.'1 0.%2 *+,

)*1.*,. -$1*,> j%7 :% &%* &,,: 2%., )'&/, ,1/+ /%$%&8 /%&*1'&) 2'$$'%&) %0 56 0'-% /,$$)>

X>

R22,.), *+, $%%- '&*% *+, *.1&)0%.21*'%& )%$7*'%& '& *+, 4# *7=,> L-'& *+, $%%- =,*D,,& 8%7. '&:,< 0'&(,.

1&: *+72= 7&*'$ *+, /%$%&8 ') :')-,.),:> B+,., )+%7$: =, &% 0$%1*'&( /+7&;)>

Y>

@$1/, *+, *7=, =1/; %& '/, 1&: .,-,1* )*,-) N 1&: X 0%. *+, .,21'&'&( *7=,) %0 *.1&)0%.21*'%& )%$7*'%& 6[C

M 1&: @#9>

[>

V)'&( 1 0.,)+ *'- 0%. ,1/+ )12-$,C -'-,* Y $ %0 *+, 1--.%-.'1*, $'(1*'%& .,1/*'%& '&*% *7=,) 4#C [ 1&: M>

@'-,* ! $ %0 *+, -U]S -$1)2': 345 '&*% *+, *7=, $1=,$,: @#> c,,- *+, *7=,) %& '/,>

P>

R&/7=1*, 1$$ X *7=,) %& '/, 0%. !O 2'&7*,)> W, )7., *+, =%**%2) %0 *+, *7=,) 1., '& /%&*1/* D'*+ *+, '/,>

M>

37.'&( *+, '&/7=1*'%& -,.'%:C $1=,$ 8%7. -$1*,)> A,*.',E, *+, X ]W 52- R@BU -$1*,) 1&: $1=,$ ,1/+ %& *+,

=%**%2 %0 *+, -$1*, 7)'&( *1=$, PK[1 1) 1 (7':,> W, )7., *% 1$)% D.'*, *+, &12,)k'&'*'1$) %0 *+, 2,2=,.)

%0 8%7. (.%7- 1&: *+, $1= :18 1&: *'2, %& ,1/+ %0 *+, -$1*,)>

\>

I,1* )+%/; *+, /,$$ )7)-,&)'%&)> @$1/, *+, 0%12 +%$:,. D'*+ 8%7. *7=,) '&*% 1 D1*,. =1*+ %. +,1* =$%/;

),* 1* XQe# 0%. , R0 7)'&( 1 D1*,. =1*+ =, )7., *+, =%**%2 %0 ,1/+ *7=, ') '& /%&*1/* D'*+

*+, D1*,.>

=>6 3??/&%#+/-< #@+/" +,/ A>B)/0'.& ,/#+ ),'0C (/"%'&D (-#0/ +,/ +E$/) $#0C '. %0/6

!!>

R&/7=1*, *+, *7=,) %& '/, 0%. Q 2'&7*,)>

!Q>

B1;, 8%7. 0%12 +%$:,. D'*+ *+, X *7=,) =1/; *% 8%7. $1= =,&/+> V)'&( )*,.'$, *,/+&'F7,C -'-,* QYO$ %0 ]W=.%*+ 60.%2 *+, 8,$$%D *7=,9 '&*% *+, 4# *7=,> G'*+ 1 &,D )*,.'$, -'-,* *'-C .,-,1* *+') )*,- 0%. *+, %*+,.

N -'&; *7=,)>

!N>

R&/7=1*, 1$$ X *7=,) 1* .%%2 *,2-,.1*7., 0%. !O 2'&7*,)>

!X>

A,*.',E, *+, $1=,$$,: ]W 52- R@BU -$1*,) 1&: -'-,* *+, ,&*'., E%$72, %0 *+, 1--.%-.'1*, *.1&)0%.21*'%&

.,1/*'%& %&*% *+, 1--.%-.'1*, -$1*, 7)'&( *1=$, PK[1 1) 1 (7':,>

!Y>

V)'&( 1 0.,)+ '&%/7$1*'%& $%%- 0%. ,1/+ )12-$,C F/"< ;/.+- @$1/, *+,

-$1*,) '& 1 )*1/; '& *+, '&/7=1*%.> j%7. *,1/+'&( 1))')*1&* D'$$ /%%.:'&1*, 1..1&('&( *+, -$1*,)>

![>

B+, -$1*,) D'$$ =, '&/7=1*,: 1* NPe# 0%. ),E,.1$ :18)C 1&: *+,& *.1&)0,..,: *% /%$: )*%.1(, 7&*'$ &,


19/21

!\

+OIB U8 =23 @&11 B$ #$%82%'&(/ )R$ ($@ )%D(482%'D)&2( '$)R2GS 34$ )R$ #%2.$G3%$ B$12@,

B+, (%1$ %0 *+') -1.* %0 *+, $1= ') *% *.1&)0%.2 =1/*,.'1$ /,$$) 7)'&( *+, $'(1*'%& .,1/*'%&) 8%7 -.,-1.,: '& ),/*'%&

P>Y> 5) 1 -%)'*'E, /%&*.%$ 0%. *+, *.1&)0%.21*'%& -.%/,:7.,C 8%7 D'$$ 1$)% =, *.1&)0%.2'&( =1/*,.'1$ /,$$) D'*+ *+,

-U]S -$1)2':>

B+') -1.* %0 *+, $1= D'$$ =, /%2-$,*,: '& (.%7-) %0 0%7. 1* ,1/+ *1=$,> S& 8%7. '/,C 8%7 D'$$ 0'&: ! *7=, %0 /%2-,*,&*/,$$)> 5* 8%7. )*1*'%&C 8%7 D'$$ 0'&: X 2'/.%*7=,) %& *+, 2'/.%*7=, .1/;> R& *+, '&/7=1*%.C 8%7 D'$$ 0'&: X ]W 52-

R@BU -$1*,)>

!>

A,*.',E, *+, X ]W 52- R@BU -$1*,) 1&: $1=,$ ,1/+ %& *+, =%**%2 %0 *+, -$1*, 7)'&( *1=$, PK[= 1) 1 (7':,>

W, )7., *% 1$)% D.'*, *+, &12,)k'&'*'1$) %0 *+, 2,2=,.) %0 8%7. (.%7- 1&: *+, $1= :18 1&: *'2, %& ,1/+

%0 *+, -$1*,)> A,*7.& *+,), -$1*,) '& *+, '&/7=1*%. 7&*'$ *+,8 1., &,,:,:>

Q>

]1=,$ *+, 0%7. 2'/.%*7=,) 0.%2 *+, 2'/.%*7=, .1/; 4# 0%. &,(1*'E, /%&*.%$C [C M 1&: @# 0%. -%)'*'E, /%&*.%$

6-U]S -$1)2':9 1&: '&'*'1$ *+,2 %& *+, *%- 1&: )':, )% *+1* *+,8 /1& =, ':,&*'0',:> c,,- *+, *7=,) %& '/,

0%. *+, &,

N>

A,*.',E, *+, $'(1*'%& .,1/*'%&) *+1* 8%7 -.,-1.,: -.,E'%7)$8>

JDB1$ +:IB> B.1&)0%.21*'%& .,1/*'%& /%2-%&,&*)>

B7=,k-$1*,

$1=,$L12-$, &12,

]'(1*'%&

.,1/*'%&

#%2-,*,&*

/,$$)

J'&1$

a%$72,

4#4,(1*'E, /%&*.%$

6E,/*%. %&$89Y $ YO $ YY $

[ !"#$%&'()%) (345


(345

@#-U]S

6-%)'*'E, /%&*.%$9! $

X>

V)'&( 1 0.,)+ *'- 0%. ,1/+ )12-$,C -'-,* Y $ %0 *+, 1--.%-.'1*, $'(1*'%& .,1/*'%& '&*% *7=,) 4#C [ 1&: M>

@'-,* ! $ %0 *+, -U]S -$1)2': 345 '&*% *+, *7=, $1=,$,: @#> c,,- *+, *7=,) %& '/,>

Y>

V)'&( 1 0.,)+ *'- 0%. ,1/+ )12-$,C F/"< ;/.+-


20/21

QO

M>

V)'&( 1 0.,)+ '&%/7$1*'%& $%%- 0%. ,1/+ )12-$,C F/"< ;/.+- @$1/, *+,

-$1*,) '& 1 )*1/; '& *+, '&/7=1*%.> j%7. *,1/+'&( 1))')*1&* D'$$ /%%.:'&1*, 1..1&('&( *+, -$1*,)>

\>

B+, -$1*,) D'$$ =, '&/7=1*,: 1* NPe# 0%. ),E,.1$ :18)C 1&: *+,& *.1&)0,..,: *% /%$: )*%.1(, 7&*'$ &,

+O+ T&*,.

8%7. -.,:'/*'%&) '& *1=$, PKP>

1>

3% 8%7 , V&D.1- *+,2 1) 27/+ 1) &,/,))1.8 *% 21;, 8%7. %=),.E1*'%&)> j%7

)+%7$: )*.'E, *% ;,,- *+, -$1*,) /$%),:>

N>

J%. ,1/+ -$1*,C .,/%.: *+, 1&)D,.) *% *+, 0%$$%D'&( '& B1=$, PKP>

1>

3%,) ,1/+ -$1*, +1E, &% (.%D*+C :')/.,*, /%$%&',) %. 1 $1D&d

=>

R0 *+,., ') /%$%&8 (.%D*+C /%7&* *+, &72=,.> 5 (.,1* &72=,. %0 /%$%&',) /1& =, ,)*'21*,: 1)

8%7 :': '& ),/*'%& [>Q>

4%*,^ R* ') -%))'=$, *+1* )1*,$$'*, /%$%&',) 218 (.%D 7)'&( *+, $'(1*'%& 2,*+%: '& *+') -.%*%/%$> #%7&* %&$8 *+,$1.(, /%$%&',)C &%* *+, *'&8 /%$%&',) D+'/+ )7..%7&: $1.(,. /%$%&',)>


21/21

Q!

JDB1$ +:+> B.1&)0%.21*'%& .,1/*'%& .,)7$*)>

@$1*,

$1=,$@.,:'/*,: (.%D*+

U.%D*+

#%$%&',)C $1D& %.

&% (.%D*+

#%$%&8

/%7&*k,)*'21*,

4#

[

M

@#

+O+ P$.)&2( %$N&$@

!> L+%7$: 8%7 , #%2-1., -$1*,) [ 1&: M D'*+ -$1*, @# 6-%)'*'E, /%&*.%$ D'*+ *+, '&*1/* -U]S -$1)2':9> I%D ') *.1&)0%.21*'%&

,00'/',&/8 100,/*,: =8 $'(1*'%&d

N> #%2-1., -$1*,) [ 1&: M> G+'/+ -$1*, (1E, 2%., /%$%&',)d @.%E':, 1 0,D ,

Documents

Supplementary Cloning Lab