Molecular Biology Experiment

 Title  Title: Size Analysis of Recombinant Plasmids Isolated by a Rapid Miniprep Procedure confirm biochemically that the transformants created in experiment 10 contain Aim: To confirm  plasmid DA! and that plasmid size is consistent "ith "ith that expected! "hich is determined by a#arose #el electrophoresis$ Abstract: The completion of a series of fe" simple steps resulted in the isolation of plasmid

DA from cellular proteins! proteins! lipids! and plasmid DA$ The %Rapid Miniprep& method bein# the  procedure of choice employed$'It "as chosen because plasmid DA needed to be isolated from the cell and this method is a (uic) and *ery efficient one$ +ntry "as made into the bacterial cell usin# lysozyme after "hich the cell contents ,proteins! lipids- "ere dissol*ed usin# SDS and  plasmid DA denatured usin# a./$ urther treatment "ith ammonium acetate! isopropanol! ethanol and a solution of TRIS/23! p/4$0! 1 mM +DTA! +DTA! 10u#5ml Rase resulted in the isolation of plasmid DA$'After completion of the experiment an electrophoretic dia#ram "as obtained that pro*ed that the plasmid "as not isolated successfully$ /ence! the experiment can be considered unsuccessful on the experimenter6s part$Therefore! reference "as made to another sample$ 7hen a plasmid is isolated and an electrophoretic anal ysis is carried out *arious topolo#ical forms of DA is obser*ed8 they are the supercoiled! linear and nic)ed5relaxed conformations$ T"o T"o topolo#ical forms "ere obser*ed for the sample sa mple ,linear and supercoiled-8 only a supercoiled conformation of the recombinant reco mbinant ,pTrc99A cys:- "as obser*ed "hereas both linear and supercoiled topolo#ies of the *ector ,pTrc99A- "ere obser*ed$ The *ector has an expected size of ;1<=bp and an actual size of approximately '>00bp "hereas the recombinant had an expected size of <9<4bp and an actual size of o*er 10000bp$'Therefore! it is (uite e*ident that the plasmid extracted "as the *ector $

Introduction : The minipreparation is a simple! ho"e*er! efficient method of isolatin#  plasmid DA from the cell$ It utilizes *arious chemicals at each step in the procedure to obtain a desired result$ The first step in the procedure in*ol*ed theuse of #lucose and the chemicals Tris /23 and +DTA$ +DTA$ ?lucose acts to maintain osmotic pressure and the Tris buffers the cell at a p/ of 4$0$ +DTA binds to di*alent metal cations in the lipid bilayer! "hich "ea)ens the cell en*elope$1The next chemicals that are used are a./ and SDS$ ao/ causes cell lysis "hereas SDS deter#ent dissol*es the lipid components of the cell membrane and cellular proteins$ Sodium hydroxide also denatures both the chromosomal andplasmid DA into sin#le strands8 the t"o strands of intact plasmid DA remainintert"ined$ Ammonium Ammonium acetate is then added to  brin# the p/ to neutrality and the DA strands can renature$ The lar#e chromosomal strands strands cannot rehybridize perfectly thou#h! ho"e*er! instead they become a partiallyhybridized tan#le$ Ammonium acetate precipitates the SDS ,"ith its lipids and proteins- from the solution$ The SDS5lipid5protein precipitate traps the tan#led chromosomal DA$1This creates the %"hite #oop& that pellets ha*e after centrifu#ation$1 .nly the plasmid DA! small fra#ments of chromosomal

DA and RA remain in solution$ Isopropanol "hich is added next rapidly precipitates nucleic acids$ /o"e*er! if allo"ed to sit for lon#er! proteins "ill also precipitate$ The DA is then "ashed "ith ethanol$ An ethanol "ash helps remo*e salts and any remainin# SDS as these can interfere "ith a restriction di#est$ The final step in the procedure "hich is to suspend the DA into Tris/23 and +DTA is done because Tris buffers the DA solution$ 1 +DTA binds di*alent cations ,especially M#@@ ions- that are a needed cofactor for bacterial nucleases and thus limits DA de#radation$ After isolation of the plasmid occurs it is analyzed by #el electrophoresis$ A#arose #el electrophoresis is a method of separatin# DA fra#ments based on size and bein# able to *ie" them$>This techni(ue is based on the )no"led#e that DA is ne#ati*ely char#ed at neutral p/ to its phosphate bac)bone$ :ecause of this fact "hen an electric potential is placed on the DA it "ill mi#rate to"ards the positi*e pole$ It is also used to #i*e the sizes of DA fra#ments after the procedure is complete$>

Method: As seen in +xperiments in Molecular :iolo#y :iochemical Applications! Pa#es 1'<1'9$ 2han#es made to the procedure 1$ In step 9 the sample "as centrifu#ed for 10mins instead of Bmins and at room temperature instead of in the cold room$ >$ In step 1B the DA pellets "ere suspended in >0ul of Tris/23 instead of B0ul$

Discussion Plasmids are circularized strands of DA found in a bacterial cell that is separate from the chromosomal DA that is present and replicates independently of the hostCs chromosomal DA$ A recombinant plasmid is a plasmid that has been clea*ed at a specific site and a DA se(uence is introduced into the clea*a#e site to form a recombinant  plasmid5dna$Plasmids can be easily isolated from the bacterial cell usin# a method such as the %Rapid Miniprep& procedure$ In order to be sure that successful isolation of the plasmid DA "as accomplished the plasmid "as run on an electrophoretic #el$ After runnin# the plasmid on the #el an electrophoreto#ram "as obtained$ This indicated "hether or not any plasmid "as isolated and if so the size and topolo#ical forms of the plasmid$Thus!it #i*es an indication of "hether or not the experiment "as successful$

There are three topolo#ical forms of DA! namelysupercoiled! nic)ed and linear$ These three topolo#ies are of different sizes and accounts for "hy one fra#ment of the DA tra*els further than the other$ The supercoiled DA is the fastest mo*in# topolo#ical form of dna of the uncut  plasmid$ The supercoiled DA has a *ery compact structure and for this reason is the fastest mo*in# conformation in the #el$ This is because the a#arose #el is of a matrix form hence the dna has to mo*e throu#h the matrix$ or this reason it is safe to say that the band that tra*elled the farthest "as the supercoiled conformation$ Another conformation of DA is the nic)ed ,relaxedconformation$ This may occur "hen topoisomerase nic) on strand of the DA helix so that DA  polymerase has access to DA for replication$ .nce this happen the super helical tension relaxes and ti#htly"ound ball becomes a floppy circle$ A nic) may also occur durin# the isolation of the  plasmid because of mechanical shearin# of the DA$ or this reason the nic)ed circle is the slo"est conformation of uncut DA$ The last conformation of DA is linear DA "hich is  produced "hen a restriction enzyme cuts a plasmid only at one site$ It can also occur because of endonuclease contamination of the isolated plasmid! or because of mild treatment$ The linear DA "ill run bet"een the supercoiled and nic)ed conformations on a # el ,possibly closer to the supercoiled band-$ .n obser*ation of the electrophoreto#ram obtained it is (uite e*ident that t"o bands "ere obtained$ /o"e*er! the t"o bands obtained "ere *er y faint in colour$ Also they barely mo*ed do"n the #el$ Thus! it is (uite ob*ious that this result cannot be usedbecause it "as not done  properly8 a *ariety of reasons may be the cause of this result obtained$ It may ha*e to do "ith the concentration of dna loaded$ An insufficient (uantity of the concentration of the dna bein# loaded on the #el mi#ht ha*e been the cause$; Increasin# the amount of dna or ensurin# that the correct *olume of dna "as ta)en up mi#ht sol*e this problem$ Another reason is that the dna mi#ht ha*e  been de#raded by nucleases$; More care should be ta)en "hen carryin# out the experiment to a*oid contamination of the dna ,a*oid touchin# e*erythin# that contact is made "ith-$ Smearin# of the dna "as (uite e*ident on the electrophoreto#ram obtained$ arious factors may cause smearin# to occur$ It may be because too much dna "as loaded on the #el$ Decreasin# the amount of DA used mi#ht sol*e this problem$;A#ain! it may be that the DA has been de#raded by nucleases$ Another reason that causes smearin# to occur is that of contamination due to protein$; This problem can be sol*ed by ensurin# that after addin# isopropanol the solution is not allo"ed to stay for too lon#er before mo*in# on to the next step as this "ill also cause precipitation of the protein to occur$ The fact that the result obtained "as unsuccessful meant that reference had to be made to a successful one$ Therefore! reference "as made to lane A4 sample :$ In this lane t"o topolo#ical forms of DA "ere obser*ed8 they "ere supercoiled and linear$ In order to determine "hich  plasmid "as obtained! the size of this plasmid "as compared to the size of the *ector ,pTrc99Aand recombinant plasmid ,pTrcp99A topAcys:-$.nly one form of the recombinant "as clearly *isiblethis "as considered to be of the linear topolo#ical form based on the distance mo*ed$ /o"e*er! there "ere t"o *isible topolo#ical forms of the *ector e*en thou#h three bands "ere

seen$ The bands "ere of the linear and supercoiled form$ The one ,*ector or reco mbinant- that most closely matches in size to the sample meansthat it "as that plasmid that "as obtained$ The expected size of the *ector "as ;1<=bp and that of the recombinant plasmid "as <9<4bp'$ /o"e*er! these "ere not the experimental sizes obtained$ The size obtained for the *ector "as approximately '>00bp and that for the recombinant "as abo*e a little abo*e the 10)b ladder$ The fact that the expected size of the *ector "as ;1<=bp and the size obtained "as '>00bp means that the plasmid mi#ht ha*e been smaller than one thou#ht and so "ould mo*e a #reater distance do"n the #el in its supercoiled conformation than one "ould expect$ /o"e*er! on the other hand! for the recombinant "ith an expected *alue of <9<4bp and an experimental *alue #reater than 10000bp means that the plasmid did not mo*e as far do"n the #el as one "ould expect$ or the sample that "as run also! an approximate size of ''00bp "as obtained$ :ased on all the sizes obtained it is (uite e*ident that the plasmid obtained is the *ectorpTrc99A ,'>00bp *s$ ''00bp-$ 3imitations that may ha*e aroused in this experiment may include

References 1$ http55"""$pps)$usm$my5lecturers5mra*i5PDEIles5Plasmidextraction>00>$pdf  Date of retrie*al >;5095>01' >$ http55faculty$plattsbur#h$edu5donald$slish5+lectrophoresis$html Date of retrie*al >;50951' '$ Fachary $ :urton$ ,199<-$+xperiments in Molecular :iolo#y :iochemical Application! Size of pTrc99A and pTrc99A cys: pa#es ;4 and 1; '$ +xperiment 1>A pa#es 1'<1'9$

;$ http55bio$classes$ucsc$edu5bio>035info5content5molbio>5molbio15troub$htm Date of retrie*al >;50951'

Guestions 1$ Did you obtain recombinants of pTrc99A containin# the topA #eneH 7hat e*idence do you ha*e to support your conclusionH  o recombinants of pTrc99A containin# the topA #ene "ere obtained$ irst of all because no  bands "ere obtained on the electrophoreto#ram and the sample that "as referred to! althou#h ha*in# DA fra#ments of different topolo#ies8 none of the topolo#ies matched the recombinant  plasmid in size$ They "ere instead of similar size to the *ector8 it "as therefore! concluded that no recombinants of pTrc99A containin# the topA #ene "as obtained$

>$ Do the transformants of 31:lue all contain the topAcys: fra#ment in pTrc99AH The transformants of 31:lue did not all contain the topAcys: fra#ment in pTrc99A because the fra#ments obtained "eren6t ali#ned "ith the control$ If the fra#ments had the #ene then they "ould be of similar size to that of the control and "ould ha*e mo*ed similar distances on the #el$ /ence it is safe to say that the 31:lue transformants did not ha*e the topAcys: fra#ment in  pTrc99A$

I "ill ma)e a #uess that you are as)in# about the restriction enzyme buffer$ Ta)e one step bac)$ 7hen e*aluatin# DA! it is common to use a restriction endonuclease "hich ma)es *ery specific cuts alon# specific palindromic se(uences on the DA$ The restriction di#ested DA is then subJect to #el electrophoresis$ The restriction endonucleases are enzymes and li)e all enzymes has an optimum temperature and salt concentration at "hich they "or)$ Restriction enzyme buffers ha*e the appropriate salts and sometimes include essential cofactors that allo" the enzyme to function$ If you "erenCt to use the appropriate buffer! chances are the enzyme didnCt "or)! the DA didnCt cut appropriately and your conclusions about the DA you use "ill be "ron#$

7hen "or)in# "ith DA samples! youCll often need to di#est them "ith restriction enzymes! meanin# youCll add restriction enzymes to ma)e cuts in the DA$ The )ind of restriction enzymes youCll typically use are type II restriction enzymes! "hich ma)e cuts at specific sites$ These restriction enzymes "ill be added to#ether "ith a restriction buffer! "hich contains in#redients that ensure the restriction enzyme "ill "or) properly$

Read more http55"""$eho"$com5infoE4B0=10;Efunctionrestriction  buffers$htmlKixzz>#/SJil'l