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Molecular Genetics 7 777120 Lab 7 Objectives Understand the principles and applications of restriction enzymes and restriction digestion

n of DNA Understand the principles and applications of DNA fragment (insert & vector) purification Understand the principles and applications of DNA ligation Understand the principles and applications of low melting temperature agarose gel electrophoresis Introduction This week you will e setting up a ligation of two DNA fragments! an insert DNA and vector DNA" DNA fragments are generated using restriction enzymes# which cut the sugar phosphate ack one at a specific ase se$uence" Two fragments thus generated can e %oined together as log as their ends are complementary" The two molecules %oin together via hydrogen onding etween the complementary ases# and DNA ligase repairs the sugar phosphate ack one y recreating the phosphodiester linkages" This week you will generated fragments for ligation and set up a ligation reaction" &nformation in your te't ook! (lasmid vectors ,loning strategies -am da () DNA /estriction enzymes! 2el electrophoresis! pp )*+)) ,hapter ) pp ).+.. pp 01+0. pp 33+34

Part A: Restriction Digestion o !ector Materials pU,56 or pU,57 DNA# uncut 8/oche Applied 9ciences : 10745782001]: (14* ng;-) 5*< /estriction uffer = 8/oche Applied 9ciences> Hind&&& restriction enzyme (5* units; -) 8/oche Applied 9ciences : 10656313001] 9terile distilled water ?icrofuge test tu es# 5"4 ml ?icrofuge test tu e trays Ad%usta le micropipettes# *+1 Ad%usta le micropipettes# 1@1* Ad%usta le micropipettes# 1*@5*** 9terile pipette tips in racks for each type of ad%usta le micropipette ?icrofuges Met"od #$or%ing in grou&s o t"ree' Aou are to digest the vector pU,56 or pU,57 (depending on what has een provided) 5" Briefly spin the tu es containing Buffer =# Hind&&&# pU, DNA in a microfuge to get all the li$uid to the ottom of each tu e"

1" 9et up the restriction enzyme reaction as follows! pU, DNA (5g;0-) 9terile distilled water 5*' uffer = =ind&&& (5*U;-) 051 1 1 -

((((((((((((((((((((((((((( )otal 20 L (((((((((((((((((((((((((((

3" Cnsure the tu e lids are closed properly and flick tu es to mi' contents" 0" Briefly spin tu es (4 sec) in microfuge (ensure tu es are alanced) to ring contents to the ottom of the tu e" 4" &ncu ate in 3.o, warm room for 3* minutes" )" Dhile your digest is incu ating# pour your agarose gel @ (art B" ." Dhile your digest is incu ating# prepare your sample! take a tu e containing 5**- of Hind&&& DNA and add 1*- of sample loading uffer" 9pin riefly to ring contents to the ottom of the tu e and leave in tu e rack until you are ready to load gel" 6" Dhen your digest is ready add 4- sample loading uffer to your pU, DNA digest" 7" Briefly spin tu es (4 sec) in microfuge (ensure tu es are alanced) to ring contents to the ottom of the tu e"

Part *: Agarose Gel +lectro&"oresis &n this section you are to pour a different type of agarose gel to what your have poured efore" Aou will pour a *"6E low melting temperature agarose gel made up in 5< TAC uffer" &t will therefore e run in 5< TAC" Materials -ow melting temperature agarose 5< TAC electrophoresis uffer# )* m- per gel tank for making up gel Cthidium romide (5* mg;m-) 9terile5**m- measuring cylinders 9terile14*m- measuring cylinders ?icrowave 5< TAC electrophoresis uffer# 14* m- per gel tank for running gel ?ini agarose gel electrophoresis tanks (ower supplies 9ample loading uffer# 3* microfuge tu es ' 5** ?icrofuge test tu es# 5"4 m-

?icrofuge test tu e trays 9afety glasses Hind&&& DNA (4*ng;-) in sample loading uffer Fne lunch o' per mini agarose gel A solute Cthanol in a wash ottle 9terile scalpel lades Met"od #$or%ing in grou&s o t"ree' 5" Deigh out ,g of low melting temperature agarose powder into a weigh oat and transfer to a sterile 14*ml flask" 1" (our in )* ml 5"*< )A+ electrophoresis uffer" 3" ?elt agarose in microwave" CAUTION: Do not allow agarose solution to boil over. If you make a mess in the microwave you must clean it u yourself before you can rocee! 0" Allow the agarose to cool down to a temperature at which you can hold the flask reasona ly comforta ly in your hands" 4" Assem le gel rig;tray and com ready for the agarose to e poured in! using masking tape# tape together two end wells of a $ide-toot"ed com (this giant well will now e your lane :5)" Use this com then as you would normally" Tape the gel tray as normal" 6. Add 5 - ethidium romide to cooled agaroseG swirl gently to mi'" .A/)IO0: "thi!ium bromi!e is a mutagen. "nsure you are wearing gloves an! safety glasses when !is ensing ethi!ium bromi!e an! ouring your gel. ." (our into assem led gel rig;tray in one movementG remove any air u tip as you have een shown" 6" -eave gel to set" .A/)IO0: To avoi! creating bubbles or ri setting. les# !o not touch gel rig$tray while gel is le using a pipette

The gel will take longer to set than the others you have oure!. 7" Fnce gel has set# remove tape and com and pour 5< TAC into gel rig until the gel is %ust su merged" 5*" -oad the samples as follows! -ane 5(taped lanes)! 51*- Hind&&& (14*ng;-) in sample loading uffer -ane 1! no sample -ane 3! no sample -ane 0! 1*- Hind&&& size marker in sample loading uffer (4*ng;-) -ane 4! no sample -ane )! no sample

-ane .! 14- pU, Hind&&& in sample loading uffer (i"e" the digest you set up earlier) 55" /un the gel at 5** H# constant voltage# until the romophenol lue is appro'" 5cm from the end" 51" (lace 2lad wrap on the UH transilluminator" Take your gel to the UH transilluminator and take a photo" 13. Aour lecturer will cut the pU, and 1k Hind&&& ands from the gel for you" =ave two tu es ready+each la elled appropriately"

Part .: D0A Ligation Aou will now ligate together your insert and vector DNAs" =ow much DNA do you haveI %indIII: Aou have run 5**- of a 14*ng; - solution of Hind&&&" This means you have run 14 g of Hind&&& DNA altogether" Aou have cut the 1"* fragment from the gel" This fragment is 5;14 of the total size of (remem er it is 4*k )J# therefore# the amount of DNA in the fragment is 5;14 of the total loaded i"e" 5 g" =ow many moles is thisI

(2) pmol 1"* k ds DNA and K 5 g ' 5*) pg;5 g ' 5pmol;))*pg ' 5;1*** K 5 ' 5#454;1*** K *".) pmol &/.: Aou digested 5ug of pU, with Hind&&& and ran all the digest on the gel" Therefore# the amount of DNA in the fragment is 5ug" =ow many moles is thisI pU, is 1")k # therefore there are slightly fewer moles of this DNA in 5g than the insert"

pmol pU, ds DNA and

K 5 g ' 5*) pg;5 g ' 5pmol;))*pg ' 5;1)6) K 5 ' 5#454;1)6) K *"4) pmol

De generally set up ligations with a 3!5 ratio of insert!vector" Lor this e'ercise# we will assume that the insert & vector are appro'" the same size" Therefore# we will add 3+0 times as much insert as vector" Materials 9terile# distilled water per ligation 5*' ligase uffer T0 DNA ligase DNA fragments in agarose# prepared in (art B .**, water ath" 5"4 m- sterile microfuge tu es ?icrofuge tu e rack Ad%usta le micropipettes# *+1 Ad%usta le micropipettes# 1@1* 9terile pipette tips in racks for each type of ad%usta le micropipette Met"od #1or%ing individuall2' 5" (lace your two tu es containing your fragments in agarose in a floaty and place in the .**, water ath" -eave the agarose to melt" 1" &n the meantime# la el two microfuge tu es MAN & MBN" 3. Add 1- of 5*' ligase uffer and 1 - of sterile# distilled water to each tu e" 4. Dhen the agarose has melted# add 3 - of pU, DNA to each tu e 5. Add 51 - of the 1"*k fragment to tu e MAN" 6. Add 51 - of sterile distilled water to tu e MBO 7. Add 5 - of T0 DNA ligase to each tu e" 6" Llick the side of the tu es and riefly spin in microfuge to ring contents to the ottom" 7" 2ive your tu es to your lecturer" 9he will put them all into a rack and incu ate them at room temperature for 3 hours" 5*" After 3 hours the tu es will e frozen at @1* *,"

Ligation
The o %ect of molecular cloning is to insert DNA from one organism into a vector that can e grown in acteria or another appropriate host" This discussion will focus on the host Escherichia coli. The most commonly used type of vector is a plasmid" These are dou le+stranded circular# e'trachromosomal DNA molecules" They carry anti iotic resistance genes for the selection of cells that have taken up the plasmid"

There are several steps in cloning a piece of DNA! 5" 2enerate a DNA fragment and a vector with compati le sticky ends o /estriction digestion o 9hearing o (,/ 1" &solate;purify the DNA fragment to e cloned o Agarose gel electrophoresis o (henol;chloroform e'traction 3" -igate fragment and vector together o DNA ligase o /e$uires energy provided y AT( to make a new phosphodiester ond 0" Transform into E.coli o acterial cells must e made PcompetentO for DNA uptake @use ,a,l 1 o ,a,l1 make plasmid DNA stick to outside of acterial cells o =eat shock is then used to create pores in the cell mem rane @ the plasmid can then enter cell 4" 9election of transformed E.coli o Bacteria are plated onto nutrient medium containing anti iotic @ plasmid vector provides resistance to this anti iotic o &f plasmid carries the lacQ gene# acteria can e selected y lue;white selection" Bacteria are plated onto nutrient medium that also contains &(T2 and <+gal" ,ells that have the plasmid carrying the insert grow as white colonies" ,ells that do not carry the insert in the plasmid grow as lue colonies" )" ,onfirmation of clone o Fnce the correct acterial colony has een identified# the cells are grown in li$uid roth o (lasmid DNA is isolated from culture i"e"OminiprepO DNA is prepared o (lasmid DNA is checked with restriction enzymes -igation is an inefficient procedures" &n ligation# the sticky ends of the vector can religate to each other# so that an insert DNA cannot ligate to the vector molecule" Thus can e minimised y having a large amount of insert DNA relative to vector" Another approach is to remove the 4Nphosphate groups from the vector using an alkaline phosphatase so that the vector DNA canNt ligate to itself" The insert DNA remains phosphorylated# there y providing 4Nphosphate groups for ligation" By this means the ligation of insert to vector is favoured" Transformation is also inefficient" Fnly a few acterial cells are transformed with plasmid" Dithin a culture of millions of acterial cells# only a few hundred are likely to end up taking up any plasmid DNA @ whether it has an insert or not" &n order to find those cells that have taken up plasmid and prefera ly recom inant plasmid# a selection procedure is re$uired" =ence the use of anti iotics and# if appropriate# lue;white selection" These methods reduce the num er of acterial colonies that have to e checked for plasmid DNA"

Re erences: 5" 2arey R/# Brown 9/# ?arkham --# Anthony /A" 2enetics -a oratory ?anual 1nd Cdition (1***)" University of Llorida# Tampa" 1" http!;;www"roche+applied+science"com;la fa$s; downloaded August# 1**4"