Laboratory 3 Protocol BMS1062 2021 PDF

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Laboratory 3 protocol BMS1062 2021...

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BMS1 0 6 2Mo l e c u l a rBi o l o g y

La bor a t or y3 Gr e e nt oBl u ePa r t 1| Pl a s mi dI s o l a t i o na n dDi g e s t I n t r od uc t i o n DNA i st h eg e n e t i cma t e r i a lc o mmo nt oa l lc e l l u l a rl i v i n go r g a n i s ms .Th ee l u c i d a t i o no fi t sf u n d a me n t a l s t r u c t u r e , t h ed o u b l eh e l i x , b yWa t s o n , Cr i c ka n dF r a n k l i ni nt h e1 9 5 0 ' so p e n e dt h ewa yf o rt h ed e v e l o p me n t o f t h efi e l do f mo l e c u l a rb i o l o g y–t h es t u d yo f i n d i v i d u a l g e n e sa n dt h e i re x p r e s s i o n . Th e s ed e v e l o p me n t sr e l i e d o nt h ea p p l i c a t i o no fas e r i e so fp r e p a r a t i v ea n da n a l y t i c a lt e c h n i q u e swh i c hh a v el e dt ot h ep r e s e n t u n d e r s t a n d i n go f t h ep r o p e r t i e sa n db i o s y n t h e s i so f n u c l e i ca c i d s( DNAa n dRNA)a n dp r o t e i n s . Mo l e c u l a rt e c hn i qu e sc a nb ea p p l i e dt ot h ema n i p u l a t i o no fDNAmo l e c u l e st h r o u g hr e c o mb i n a n tDNA t e c h n o l o g y .T h e s et e c h n i q u e s( o f t e nr e f e r r e dt oa sge ne t i ce ng i ne e r i n g)i n c l u d et h eu s eo fr e s t r i c t i o n e n d o n u c l e a s e st oc u tDNAmo l e c u l e sa tp a r t i c u l a rs i t e s ,DNAl i g a s et oj o i nDNAf r a g me n t si nv i t r o ,a n d t r a n s f o r ma t i o nt e c h n i q u e st or e i n t r o d u c er e c o mb i n a n tDNAmo l e c u l e si n t ol i v i n gc e l l swh e r et h e yc a nb e p r o p a g a t e di nl a r g eq u a n t i t i e s . T h eu s eo f r e s t r i c t i o ne n z y me sa n dt h emo l e c u l a rc l o n i n go f DNAf r a g me n t sa r et e c h n i q u e sc u r r e n t l ya p p l i e d i nma n ymo l e c u l a rb i o l o g yr e s e a r c hl a b o r a t o r i e sa r o u n dt h ewo r l df o rt h es t u d yo fawi d er a n g eo fg e n e t i c s y s t e ms .Ma n yc l o n i n gt e c h n i q u e si n v o l v et h eu s eo ft h es ma l lc i r c u l a rDNAmo l e c u l e sc a l l e db a c t e r i a l p l a s mi d st h a tc a na c ta sc a r r i e r s( o rv e c t o r s )f o ro t h e rDNAf r a g me n t sk n o wna si n s e r t s .T h e s ep l a s mi d v e c t o r s , s u c ha sp Gl o , c o n t a i na t l e a s t o n eu n i q u e( i . e . o c c u ro n c eo n l y )r e s t r i c t i o ne n z y mec l e a v a g es i t e( i n t o wh i c hf o r e i g nDNAf r a g me n tc a nb ei n s e r t e d ) , a swe l l a so n eo rmo r ed r u gr e s i s t a n c eg e n e swh i c hf a c i l i t a t e t h eg e n e t i cs e l e c t i o no f h o s t c e l l si nwh i c ht h ep l a s mi dDNAi sr e p l i c a t i n g . p Gl oi sad o u b l e s t r a n d e dc i r c u l a rp l a s mi do f5 3 7 1b a s ep a i r s( b p ) .T h ep l a s mi dh a st h eg e n ef o rGr e e n F l uo r e s c e nt Pr o t e i n( GF P) a n dt h ed r u gr e s i s t a n c eg e n ef o ra mp i c i l l i na swe l l a sma n yo t h e rf e a t u r e st h a t we wi l l e x p l o r eo v e r t h es e me s t e r . F o r ap l a s mi dma po f p Gl os e eFi g u r e1o nt h ef o l l o wi n gp a g e . T h i sl a b o r a t o r ye x e r c i s ewi l l i n t r o d u c ey o ut ot h eh a n d l i n go f b a c t e r i a l p l a s mi d s . Y o uwi l l i s o l a t et h e mf r o mt h e i r b a c t e r i a l h o s t s , c h a r a c t e r i s et h e i r DNAb yg e l e l e c t r o p h o r e s i sa n db e g i nt oma n i p u l a t et h e mb yd i g e s t i o nwi t ha t h r e s t r i c t i o ne n z y me . ( Re f :Al be r t s , 5the d. pp5 3 2 3 5 , 5 4 0 4 2 ;6 e dpp4 6 3 4 7 3 ) .

Copyright Warning: All materials produced for teaching this course of study, including all lectures and any supplementary materials are protected by copyright. You are permitted to use these materials only for your personal study and research. Use of the materials for any other purposes, including sale of lecture notes, without the express permission of the copyright owner, may infringe copyright. The copyright owner may take action against you for infringement Page | 1

BMS1 0 6 2Mo l e c u l a r Bi o l o g y

F i gu r e1 :Ama po f t h ep l a s mi dp Gl os h o wi n gr e s t r i c t i o ne n d o n u c l e a s es i t e sa n d k e yf e a t u r e s

Copyright Warning: All materials produced for teaching this course of study, including all lectures and any supplementary materials are protected by copyright. You are permitted to use these materials only for your personal study and research. Use of the materials for any other purposes, including sale of lecture notes, without the express permission of the copyright owner, may infringe copyright. The copyright owner may take action against you for infringement Page | 2

BMS1 0 6 2Mo l e c u l a r Bi o l o g y

Pa r t1 . 1 : Pr e pa r a t i o no fp l a s mi df r omDNAf r o mt r a n s f o r me dE. c ol i Ce l l s Out c ome s De mo n s t r a t et h ec o r r e c t u s eo f mi c r o p i p e t t e s De s c r i b et h et h e o r e t i c a l b a s i sb e h i n de a c hs t e po f as p i nc o l u mn b a s e dp l a s mi dDNAi s o l a t i o n Di s c u s st h es u i t a b i l i t yo f p l a s mi dp Gl of o r l a b o r a t o r yp r o c e d u r e s I mp l e me n t mo l e c u l a r t e c h n i q u e st oi s o l a t ep l a s mi dp Gl of r o mE. c o l i De mo n s t r a t et h ea b i l i t yt oc o mmu n i c a t et h r o u g hs c i e n t i fi cr e p o r t i n g

    

Wo r ki npa i r s

Ma t e r i a l s Co ns uma bl e s :      

St e r i l e1mLb l u et i p s St e r i l e2 0 0µ Ly e l l o wt i p s St e r i l e1 . 5mLmi c r o f u g et u b e s T o o t h p i c k s Mi n i Pr e pc o l u mn Co l l e c t i o nt u b e s

Sol ut i o ns( s e eApp e nd i xf orc omp os i t i o ns ) :  E. c o l i ( wi t hp Gl o ) c u l t u r ei nL b r o t hg r o wni na3 7 ° Ci n c u b a t o r o v e r n i g h t  Re s u s p e n s i o nb u ff e r ( k e e po ni c e )  L y s i sb u ff e r  Pr e c i p i t a t i o nSa l t ( k e e po ni c e )  Bi n d i n gBu ff e r  Wa s hBu ff e r 1  Wa s hb u ff e r2  T EBu ff e r Eq ui pme nt :  

Mi c r o p i p e t t e s He r a e u sPi c o1 7mi c r o c e n t r i f u g e

T h el a b o r a t o r ys t a ffwi l l h a v es e t u pan u mb e r o f o v e r n i g h t c u l t u r e so fE. c o l i s t r a i nc o n t a i n i n gt h ep l a s mi dp Gl o i n t o4 0mLo fL b r o t ha n dAmp i c i l l i n( L BA)i n2 5 0mLc o n i c a l fl a s k s .Y o u rT e a c h i n gAs s o c i a t ewi l l a d v i s ey o u wh e nt h ec e l l swi l l b er e a d yf o rh a r v e s t i n g . Copyright Warning: All materials produced for teaching this course of study, including all lectures and any supplementary materials are protected by copyright. You are permitted to use these materials only for your personal study and research. Use of the materials for any other purposes, including sale of lecture notes, without the express permission of the copyright owner, may infringe copyright. The copyright owner may take action against you for infringement Page | 3

BMS1 0 6 2Mo l e c u l a r Bi o l o g y

Me t hod Be f or ey ous t a r t :t h e r ea r ema n ys t e p si nt h i sp r o t o c o l t h a tr e q u i r ec h a n g e si np i p e t t i n gv o l u me ,c e n t r i f u g e s p e e d sa n dt i me s . Ma k es u r ey o ua r ec o n s t a n t l yc h e c k i n gt h e s eb e f o r ee a c hs t e p . 1 .Pi pe t t et wol ot so f1 . 3mLo f t h eE. c o l i b a c t e r i a l c u l t u r ei n t ot wo1 . 5mLt u b e s( u s eami c r o p i p e t t es e t a t 6 5 0µ La n dd i s p e n s et wi c ei n t oe a c ht u b e ) . 2 .Pl a c et h e s et u b e si n t ot h eHe r a e u sPi c o1 7c e n t r i f u g ea n ds p i na t1 3 , 0 0 0r p mf o r1mi n .Ge n t l y r e mo v et h es u p e r n a t a n tu s i n gami c r o p i p e t t ewi t haf r e s ht i p ,l e a v i n gt h ep e l l e ta sd r ya sp o s s i b l e . Not e : y o u ra i mh e r ei st or e mo v ea l l o f t h es u p e r n a t a n t s oma k es u r ey o uu s ea na p p r o p r i a t ep i p e t t e s e t t i n g . Y o uc a nd os oi ns e v e r a l s t e p s . Di s c a r dt h es u p e r n a t a n t i n t ot h el i q u i dwa s t ej a r p r o v i d e d . 3 .Ad d7 5µ Lo fi c e c o l dRe s u s p e n s i o nBu ff e rt oe a c ht u b ea n dr e s u s p e n dt h ec e l l sb yv o r t e x i n g v i g o r o u s l yu n t i l n oc e l l p e l l e t i sv i s i b l e . Co mb i n et h es u s p e n s i o n st o g e t h e ri no n eo f t h et u b e s . Di s c a r d t h eo t h e r t u b e . 4 .Ad d1 5 0µ Lo fL y s i sBu ff e ra n dmi xg e n t l yb yi n v e r t i n gt h ec a p p e dt u b e5t i me s .Don o tv o r t e x . I n c u b a t ea t r o o mt e mp e r a t u r ef o r 3mi n . 5 .Ad d1 5 0µ Lo fi c e c o l dPr e c i p i t a t i o nSa l ta n di mme d i a t e l ymi xb yi n v e r t i n gt h et u b e5t i me s .Dono t v or t e x . Ce n t r i f u g ea t 1 3 , 0 0 0r p mf o r5mi n . Wh i l et h ep r e pi sc e n t r i f u g i n g , p l a c et h emi n i p r e pc o l u mn i n s i d et h e2mLc o l l e c t i o nt u b e . 6 .No wc a r e f u l l yr e mo v et h eg e l a t i n o u sp e l l e t b ys c o o p i n gi t o u t wi t ht h eb l u n t e n do f at o o t h p i c ko r y e l l o w p i p e t t et i pa n dd i s c a r di t i n t ot h eb i o l o g i c a l wa s t eb i n . 7 .Ad d6 0 0µ Lo ft h eBi n d i n gBu ff e ra n dmi xb yi n v e r t i n g5t i me s .T r a n s f e rt h ee n t i r es o l u t i o ni n t ot h e mi n i p r e pc o l u mn/c o l l e c t i o nt u b eu s i n gami c r o p i p e t t ewi t haf r e s ht i p .Not e :y o ua i mh e r ei st o r e mo v ea l l o f t h es o l u t i o nt ot h ec o l u mns oma k es u r ey o uu s ea na p p r o p r i a t ep i p e t t es e t t i n g . 8 .Ce n t r i f u g ea t2 , 0 0 0r p mf o r3 0s e c .T od ot h i s ,p l a c ey o u rt u b ei nt h ec e n t r i f u g e ,r e s e tt h es p e e dt o 2 , 0 0 0r p m, s e t t h et i me r t o‘ h o l d ’ a n dwh e ni t r e a c h e s3 0s e cp r e s s‘ s t o p ’ . 9 .Re mo v et h ec o l u mnf r o mt h ec o l l e c t i o nt u b e ,d i s c a r dt h efl o wt h r o u g hi n t ot h el i q u i dwa s t ej a ra n d r e p l a c et h ec o l u mnb a c ki n t ot h et u b e . Ad d5 0 0µ Lo f Wa s hBu ff e rNo . 1a n dc e n t r i f u g ea t 2 , 0 0 0r p m f o r 3 0s e ca sb e f o r e . 1 0 .Ag a i n ,d i s c a r dt h efl o wt h r o u g ha n dr e p l a c et h ec o l u mn .Ad d9 0 0µ Lo fWa s hBu ff e rNo .2a n d c e n t r i f u g ea t2 , 0 0 0r p mf o r1mi n .Di s c a r dt h efl o wt h r o u g hi n t ot h el i q u i dwa s t ej a r .Ce n t r i f u g ea t 1 3 , 0 0 0r p mf o r 1mi nt or e mo v ea n yr e s i d u a l wa s hb u ff e r . 1 1 .T oe l u t et h ep l a s mi d DNA f r o mt h ec o l u mn ,p l a c et h ec o l u mn i n s i d ea1 . 5 mL r e c o v e r y ( mi c r o c e n t r i f u g e )t u b e .Ad d6 0µ Lo fT EBu ff e rd i r e c t l yt ot h ec o l u mna n di n c u b a t ef o r3mi na tr o o m t e mp e r a t u r e . Ce n t r i f u g ea t 1 3 , 0 0 0r p mf o r3 0s e c . Re mo v ea n dd i s c a r dt h ec o l u mn .No t e :b es ur et o l a b e l t h i sfi na l r e c o v e r yt u bec a r e f u l l ya sy ouwi l l ne e di t o v e rt her e ma i n de ro fs e me s t e r .

Copyright Warning: All materials produced for teaching this course of study, including all lectures and any supplementary materials are protected by copyright. You are permitted to use these materials only for your personal study and research. Use of the materials for any other purposes, including sale of lecture notes, without the express permission of the copyright owner, may infringe copyright. The copyright owner may take action against you for infringement Page | 4

BMS1 0 6 2Mo l e c u l a r Bi o l o g y

Pa r t 1 . 2 :Re s t r i c t i one n z y medi ge s t i ono fi s o l a t e dpl a s mi dDNA Out c ome s De mo n s t r a t et h ec o r r e c t u s eo f mi c r o p i p e t t e s I d e n t i f ya n dd e s c r i b et h ek e yf e a t u r e so f t h ep Gl op l a s mi d , a sr e p r e s e n t e dt h r o u g hap l a s mi dma p I mp l e me n t mo l e c u l a r t e c h n i q u e st od i g e s t p l a s mi dp Gl owi t hr e s t r i c t i o ne n z y me Emp l o ya nu n d e r s t a n d i n go f p l a s mi dma p si n c l u d i n gr e s t r i c t i o ne n d o n u c l e a s es i t e st oh y p o t h e s i s et h e s u c c e s so f ar e s t r i c t i o nd i g e s t De mo n s t r a t et h ea b i l i t yt oc o mmu n i c a t et h r o u g hs c i e n t i fi cr e p o r t i n g

    

Ma t e r i a l s Co ns uma bl e s :   

St e r i l e1mLb l u et i p s St e r i l e2 0 0µ Ly e l l o wt i p s St e r i l e1 . 5mLmi c r o f u g et u b e s

Sol ut i o ns     

Pl a s mi dDNAp r e p a r a t i o n( f r o mPa r t 1 ) Ec o RI e n z y me( 2 , 0 0 0u n i t s ( U) / ml ) 1 0xEc o RI d i g e s t b u ff e r St e r i l ed i s t i l l e dwa t e r SEB( s u c r o s e EDT Ab r o mo p h e n o l b l u e ) Bu ff e r

Eq ui pme nt :   

Mi c r o p i p e t t e s He r a e u sPi c o1 7mi c r o c e n t r i f u g e He a t i n gb l o c ks e t a t 3 7 ° C

Copyright Warning: All materials produced for teaching this course of study, including all lectures and any supplementary materials are protected by copyright. You are permitted to use these materials only for your personal study and research. Use of the materials for any other purposes, including sale of lecture notes, without the express permission of the copyright owner, may infringe copyright. The copyright owner may take action against you for infringement Page | 5

BMS1 0 6 2Mo l e c u l a r Bi o l o g y

Me t hod 1 .Co mp l e t et h et a b l e sb e l o wf o rT u b e sAa n dB.Pr e p a r ee a c hmi x t u r ei n1 . 5mLmi c r o f u g et u b e sa n d k e e pt h e mo ni c e : T ub eA Pl a s mi dDNAp r e p a r a t i o n

1 5µ L

1 0xEc o RI d i g e s t b u ff e r

2µ L

St e r i l ed i s t i l l e dwa t e r t oafi n a l v o l u meo f 2 0µ L

3µ L

F i n a l Vol ume

2 0µ L

T ub eB Pl a s mi dDNAp r e p a r a t i o n 1 0xEc o RI d i g e s t b u ff e r Ec o RI e n z y me( 4u n i t s( U) ) St e r i l ed i s t i l l e dwa t e r t oafi n a l v o l u meo f 2 0µ L F i n a l Vol ume

1 5µ L 2µ L 2µ L 1µ L 2 0µ L

I MPORT ANT| Donot di s c a r dy ouror i gi na l pl a s mi dDNApr e pa r a t i on Y ouwi l l n e e di t i nL a bor a t or y5 2 .Br i e fl yc e n t r i f u g eb o t ht u b e sf o r2 0s e ca t 1 3 , 0 0 0r p m, ( s e t t h et i me rt o‘ h o l d ’ a n dwh e ni t r e a c h e s3 0 s e cp r e s s‘ s t o p ’ )t oe n s u r ep r o p e r mi x i n go f c o n t e n t s . 3 .I n c u b a t et u b e sa t 3 7oCf o r 1h o u ri nah e a t i n gb l o c k . 4 .Ad d5µ Lo f SEBBu ff e r t oe a c ht u b e . 5 .Cl e a r l yl a b e la n dt a gy o u rt u b e sa n dh a n dt h e mi na tt h ep r e pr o o mt ob es t o r e df r o z e nu n t i lt h e a g a r o s eg e l i sr u ni nL a b o r a t o r y5 .

Copyright Warning: All materials produced for teaching this course of study, including all lectures and any supplementary materials are protected by copyright. You are permitted to use these materials only for your personal study and research. Use of the materials for any other purposes, including sale of lecture notes, without the express permission of the copyright owner, may infringe copyright. The copyright owner may take action against you for infringement Page | 6

BMS1 0 6 2Mo l e c u l a r Bi o l o g y

Results Ma i n t a i n i n ga c c u r a t ea n du p t od a t el a b o r a t o r yr e c o r d sa n dn o t e si sc r i t i c a l l yi mp o r t a n t . I nr e s e a r c h l a b o r a t o r i e si t i se s s e n t i a l i no r d e r t ob ea b l et or e p o r t o nwh a t wed o , b u t i sa l s oc r i t i c a l f o r r e s e a r c hi n t e g r i t y a n dt r a n s p a r e n c y : o u rr e c o r d sa r ee v i d e n c eo f t h ewo r ka n dr e s u l t swep r o d u c e . 

Du r i n gy o u r l a b o r a t o r ys e s s i o n , u s et h i ss e c t i o nt or e c o r da n yn o t e sa n do b s e r v a t i o n sf r o mt h e e x p e r i me n t i n c l u d i n ga n ya l t e r a t i o nt ot h ep r o t o c o l t h a t we r ema d e

Not e s

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BMS1 0 6 2Mo l e c u l a r Bi o l o g y

APPENDI X COMPOSI TI ONANDUSEOFSOMESOLUTI ONSUSEDI NTHI SPRACTI CAL Re s u s p e n s i o nBu ff e r : c o n t a i n sT r i s HCl p H8 , EDT A, 0 . 1mg / ml RNa s eA L y s i sBu ff e r :

c o n t a i n sNa OH, SDS

Bi n d i n gBu ff e r :

c o n t a i n sa c e t a t ea n dg u a n i d i n eh y d r o c h l o r i d e

Wa s hBu ff e r 1 :

c o n t a i n sNa Cl , EDT A, T r i s HCl p H8

Wa s hBu ff e r 2 :

c o n t a i n sa c e t a t e , g u a n i d i n eh y d r o c h l o r i d e , EDT A, e t h a n o l

T Eb u ff e r :

1 0mMT r i s HC1p H8 , 1mMEDT A ( u s e da sag e n e r a l n u c l e i ca c i ds o l v e n t ) .

SEB:

5 0 %s u c r o s e , 0 . 1 MEDT Ap H8 , 0 . 1 %b r o mo p h e n o l b l u e ( u s e da sl o a d i n gs o l u t i o nf o r g e l s a mp l e s ) .

Ec o RI e n z y me :

2 0 0 0u n i t s / ml

1 0xd i g e s t b u ff e r f o r Ec o RI :

1MT r i s HC1p H7 . 5 , 0 . 5MNa Cl , 0 . 1MMg Cl 2 ( u s e da ss t o c kb u ff e rf o rEc o RI d i g e s t i o n s ) .

L b r o t h :

1 %y e a s t e x t r a c t , 1 %t r y p t o n e , 0 . 5 %Na Cl ( u s e da sag e n e r a l g r o wt hme d i u mf o rE. c o l i c e l l s ) .

Eb u ff e r :

4 0mMT r i s HCl , p H7 . 8 , 2 0mMNaa c e t a t e , 2mMN EDT A 2a ( u s e da se l e c t r o p h o r e s i sb u ff e r )

d H20 :

d i s t i l l e d , a u t o c l a v e dwa t e r

Copyright Warning: All materials produced for teaching this course of study, including all lectures and any supplementary materials are protected by copyright. You are permitted to use these materials only for your personal study and research. Use of the materials for any other purposes, including sale of lecture notes, without the express permission of the copyright owner, may infringe copyright. The copyright owner may take action against you for infringement Page | 8...