777120 2006 Lab 1 Student Version

8/13/2019 777120 2006 Lab 1 Student Version

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Molecular Genetics 7777120

Lab 1Objectives

Define restriction enzymes and their role in genetic engineeringUnderstand the principles and applications of restriction enzymes and restriction

digestion of DNA

Become proficient in the loading of agarose gels

Introduction

Lambda () is a virus that infects bacteria. his virus does not infect humans and is! therefore!

safe for us to study.

"n this investigation! you #ill observe the effects of three restriction enzymes on DNA. Arestriction enzyme (also $no#n as an endonuclease) is an enzyme that recognises a specificbase pair se%uence #ithin DNA and cuts the DNA at that se%uence. he enzyme #ill cut everytime the se%uence it recognises appears #ithin the DNA molecule. he DNA fragmentsgenerated are called restriction fragments. hese fragments can be separated from each otheraccording to their size by gel electrophoresis.

he restriction enzymes you #ill use are Eco&"! Pst" and Hind""". Ne't #ee$! you #ill use gelelectrophoresis to separate the resulting DNA pieces.

"nformation in your te'tboo$

Lambda () DNA pp *+** &estriction enzymes pp ,-+,*

&estriction mapping pp ,*+,

/el electrophoresis pp 00+01

Part A: Agarose Gel Electroporesis

Materials

#o large 23 agarose4 5.16 B7 gels5.16 B7 electrophoresis buffer! -L per large gel tan$ for running gel

ris47DA buffer (7) p8 .5! 05 microfuge tubes ' 255L

9ini agarose gel electrophoresis trays9ini agarose gel electrophoresis combs9ini agarose gel electrophoresis tan$s:o#er supplies;terile -15ml flas$sAgarose po#der


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7thidium bromide (25 mg4mL)

;ample loading buffer! 05 microfuge tubes ' 255L

9icrofuge test tubes! 2.1 l

9icrofuge test tube trays;afety glasses

Metod A!1 "#o be done individuall$%

=ou have been provided #ith t#o 23 agarose gels already made up. =ou are to use these gelsto practice loading samples. =ou #ill need to ta$e turns #ith others in the class.

2. a$e three 2.1 mL and place into a microfuge test tube tray.

2. :ipette 1L of sample loading buffer into each tube.

3. :ipette 21L of 7 buffer into each tube.

,. 7nsure the tube lids are closed properly and flic$ tubes to mi' contents.

1. Briefly spin tubes (1 sec) in microfuge (ensure tubes are balanced) to bring contents to

the bottom of the tube.

6. :ipette 2L of 7 buffer into each of three #ells on one of the gels provided. =our

lecturer #ill demonstrate ho#.

Metod A!2 "&or' in groups o( tree%

=ou #ill learn ho# to pour an agarose gel.

CAUTION: WEAR GLOVES

2. you lecturer #ill sho# you ho#.

CAUTION: Do not allow agarose solution to boil over I! "ou #a$e a #ess in t%e#i&rowave "ou #ust &lean it u' "oursel! be!ore "ou &an 'ro&ee(,. Allo# the agarose to cool do#n to a temperature at #hich you can hold the flas$

reasonably comfortably in your hands.

1. Assemble gel rig4tray and comb ready for the agarose to be poured in.

6. Add 2L ethidium bromide to cooled agarose? s#irl gently to mi'.

)A*#IO+: Et%i(iu# bro#i(e is a #utagenEnsure "ou are wearing gloves an( sa!et"glasses w%en (is'ensing et%i(iu# bro#i(e an( 'ouring "our gel

*. :our into assembled gel rig4tray in one movement? remove any air bubble as sho#n by

your lecturer.

. Leave gel to set.

)A*#IO+: To avoi( &reating bubbles or ri''les) (o not tou&% gel rig*tra" w%ile gel issetting

@. nce gel has set! remove comb and pour -15mL 5.16 B7 into gel rig.


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25."f there is time! repeat method A.2 using your o#n gel and t#o microfuge tubes. =ou #ill

have #ells to load samples into. 7ach of the three in your group #ill have t#o #ells into

#hich you can practice loading a sample.

Part ,: -estriction .igest

Materials

Hind""" restriction enzyme (25 units4 L) &oche Applied ;ciences C 10656313001] 25

microfuge tubes ' 1L

Pst" restriction enzyme (25 units4 L) &oche Applied ;ciences C 10621625001] 25 microfuge

tubes ' 1L

Eco&" restriction enzyme (25 units4 L) &oche Applied ;ciences C 10703737001] 25

microfuge tubes ' 1L

256 &estriction buffer 8 &oche Applied ;ciences 25 microfuge tubes ' -5uL

256 &estriction buffer B &oche Applied ;ciences 25 microfuge tubes ' -5uLLambda DNA! uncut &oche Applied ;ciences C 10745782001]: (-15 ng4L)

9icrofuge test tubes! 2.1 ml9icrofuge test tube trays

AdEustable micropipettes! 5+- L

AdEustable micropipettes! ->-5 L

AdEustable micropipettes! -5>-55 L

;terile pipette tips for each type of adEustable micropipette9icro#ave oven9icrofuges

Metod ,!1: "&or' in groups o( tree%

=ou are to set up si' restriction digests of (Lambda) DNA. 7ach digest #ill be set up in a

separate tube.

2. a$e si' 2.1 ml microfuge tubes and label each tube #ith your name and each tube as

FAG! FBG FHG! FDG! F7G and FIG. 8old your tubes in a microfuge tray.

-. ;et up the restriction enzyme reactions by adding each of the ingredients listed belo# for

each tube. Add the reaction ingredients in the order listed.

#ube .+A"2/0ngL%

teriledeionised

&ater

10,u((er

3

10,u((er

,

E&o-I

"10*L% +stI"10*L% ,indIII"10*L%A ,L 20L -L + 2L + +

B ,L 20L -L + + 2L +

H ,L 20L + -L + + 2L

D ,L 2-L -L + 2L 2L +

7 ,L 2-L -L + 2L + 2L

I ,L 2-L -L + + 2L 2L


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0. 7nsure the tube lids are closed properly and flic$ tubes to mi' contents.

,. Briefly spin tubes (1 sec) in microfuge (ensure tubes are balanced) to bring contents to

the bottom of the tube.

1. "ncubate overnight in 0*oH #arm room.


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-estriction En4$5es

e6uence recognition cleavage:he names of the enzymes are derived from the bacterium from #hich they #ere isolated.

e.g. Eco&" is purified from Escherichia coli. Bam8" is purified from a Bacillusamyloliqueraciens.

"n some cases! more than one restriction enzyme can be purified from the same organism."n this case! the enzyme names have different numerals after the bacteriumGs name.

e.g. Pvu" and Pvu"" are different restriction enzymes from the same organism!Proteus vulgaris.

he most useful restriction enzymes recognise ,+ base pair sites! of symmetric invertedrepeats! called palindromes. "n the e'ample! Eco&"! notice ho# the 1G to 0G se%uence ontop J bottom strands is identical

Eco&" 1G+/AAH+0G0G+HAA/+1G

;ome restriction enzymes cut both DNA strands at the same position! creating blunt+ended fragments. 8o#ever! the maEority of enzymes ma$e cuts staggered on eachstrand! resulting in a fe# base pairs of single+stranded DNA at each end of the fragment!$no#n as Fstic$yG ends. hese #ill either be 1G overhangs or 0G overhangs. Hleavageresults in either a 1G overhang! 0G overhang or a blunt end.

o Hommonly used enzymes that give a 1G overhang Eco&"! Bam8"!Xba"! Hind"""

o Hommonly used enzymes that give a 0G overhang Pst"! Kpn"! Pvu"! Sac"

o Hommonly used enzymes that give a blunt end Sma"! Eco&K

&estriction enzymes #ith shorter recognition se%uences cut more fre%uently than those#ith longer recognition se%uences. Ior e'ample! a , base pair (bp) cutter #ill cleave! onaverage ,,(-1) bases! #hile a bp cutter cleaves every ,(,5@) bases.

;ome enzymes have degenerate recognition se%uences! i.e. they can recognise andcleave several similar but different sites. Ior e'ample

o Ava"" cuts at //AHH as #ell as //HH.

o Ava" cuts at H:yH/:u/. :y stands for pyrimidine i.e. or H! #hile :u stands for

purine i.e. A or /. hus!Ava" #ill digest HH/A/! HH///! HHH/A/ and

HHH///.o Dde" cuts at HNA/! N being any bases i.e. A! H! / or . hus! it cleaves

HAA/! HHA/! H/A/! HA/

"soschizomers are different enzymes that share the same specificity! i.e. recognise thesame site. hese are derived from different bacteria.

o e.g. Sau0A is an isoschizomer of Mbo" > they both cleave the site /AH.

8o#ever! in this e'ample Mbo" does not cleave methylated DNA! #hile Sau0Adoes. Sau0A can therefore be used instead of Mbo" #here necessary. ;uchdifferences can be very useful.

o ther isoschizomers recognise a subset of another enzymeGs site. Ior e'ample!

Sau0A (/AH) #ill recognise! cleave and leave the same overhang as Bam8"(//AHH) and Bgl"" (A/AH).


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he type of restriction digest done depends on the do#nstream usage of the DNA fragments.Ior e'ample! DNA is digested #ith restriction enzymes a) for restriction mapping DNAmolecules and b) prior to cloning.

-estriction 5apping"t is possible to determine the relative positions of a variety of restriction sites in a fragment ofDNA #ithout se%uencing it. his process is called restriction mapping. o ensure the greatest

accuracy! a large panel of restriction enzymes are generally used to digest the DNA fragment ofinterest. After determining #hich enzymes actually cut! the restriction sites #ould then beordered to generate a linear map. his is largely a trial+and+error process! so the more enzymesused! the more accurate the restriction map #ill be. An e'ample restriction map for the plasmidpB&0-- is given belo#

Iigure 2 &estriction map of the plasmid! pB&0-- (2).

)loning&estriction enzymes are essential tools for cloning fragments of DNA. 8o# they are used forthis purpose #l be discussed in more detail in a later lecture J lab class.


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the fragment you #ish to clone but is also compatible #ith the cloning site #ithin the targetvector you #ish to use.

7ach restriction enzyme is supplied as a given number of units! corresponding to a specifiedlevel of enzyme activity. 7ach supplier ships their enzyme #ith a data card that indicates theunits of activity! the concentration of enzyme! optimal reaction conditions! storage conditions!contamination! etc.

8istorically! one unit (2U) of an enzyme (i.e. the specific activity) #as defined as the amount of

enzyme re%uired to digest 2g Lambda DNA in 2 hour at its optimal temperature. his is no#

outdated since many enzymes donGt cut Lambda. Ior e'ample! the company Ne# 7nglandBiolabs defines a unit as Mne unit of restriction endonuclease activity is defined as the amount

of enzyme re%uired to completely digest 2 g of substrate DNA in a total reaction volume of

5.51 ml in one hour using the N7B buffer provided. his means that 2Unit of enzyme shouldcut AN= DNA under the conditions defined.

/enerally! ho#ever! for more comple' DNA molecules such as genomic DNA or supercoiledplasmid DNA! more enzyme is re%uired for longer than for simple molecules such as rela'edplasmids. 9ost researchers add a ten+fold e'cess of enzyme to their reactions in order toensure complete cleavage.

&estriction enzymes are supplied by the companies in glycerol. his chemical prevents theproteinaceous enzyme from freezing #hen stored at >-5oH (the optimal storage temperature). "fan enzyme froze it #ould become inactive. 8o#ever! too much glycerol in a restriction digestcan interfere #ith the enzymeGs activity. hus! the rule of thumb is not to have more than 253 ofthe total reaction volume as restriction enzyme

-eaction volu5e

9ost digests are carried out in a volume bet#een 25 and 15 OL. &eaction volumes smallerthan25 OL are susceptible to pipetting errors! and are not recommended.

#e botto5 line is9999,e(ore setting up a restriction digest cec' to 5a'e sure tat$ou are using te proper conditions;


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-e(erences

2. http44d#b.unl.edu4eacher4N;I4H54H5Lin$s4###.fermentas.com4ech"nfo4NucleicAcids4mappbr0--.htmdo#nloaded 20th Puly! -551.

-. http44###.roche+applied+science.com4pac$+insert45*50*0*a.pdf. "nformation regardingEco&"! do#nloaded 22th Puly! -551

0. http44###.roche+applied+science.com4pac$+insert451020a.pdf. "nformation regarding

Hind"""! do#nloaded 22th Puly! -551.4. http44###.roche+applied+science.com4pac$+insert45-2-1a.pdf. "nformation regarding

Pst"! do#nloaded 22th Puly! -551.
http://dwb.unl.edu/Teacher/NSF/C08/C08Links/www.fermentas.com/TechInfo/NucleicAcids/mappbr322.htmhttp://dwb.unl.edu/Teacher/NSF/C08/C08Links/www.fermentas.com/TechInfo/NucleicAcids/mappbr322.htmhttp://www.roche-applied-science.com/pack-insert/0703737a.pdfhttp://www.roche-applied-science.com/pack-insert/0656313a.pdfhttp://www.roche-applied-science.com/pack-insert/0621625a.pdfhttp://dwb.unl.edu/Teacher/NSF/C08/C08Links/www.fermentas.com/TechInfo/NucleicAcids/mappbr322.htmhttp://dwb.unl.edu/Teacher/NSF/C08/C08Links/www.fermentas.com/TechInfo/NucleicAcids/mappbr322.htmhttp://www.roche-applied-science.com/pack-insert/0703737a.pdfhttp://www.roche-applied-science.com/pack-insert/0656313a.pdfhttp://www.roche-applied-science.com/pack-insert/0621625a.pdf

777120 2006 Lab 1 Student Version

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