AP Biology Lab 6: Molecular Biology Objectives: At the end of this lab students should: Understand the principles and

d practice of agarose gel electrophoresis Demonstrate the separation of molecules based on charge Examine the rate of separation of molecules based on size Determine which component is missing in a mixture of several dyes Background oncepts in molecular biology are often difficult to grasp! "hen trying to visualize the invisible world of the molecule# students are too often confronted by abstract theory! $ut li%e everything else in the field of biotechnology# things are changing fast! &n '()*# a scientist named Oliver +mithies invented gel electrophoresis! ,el electrophoresis is an advancement in biotechnology that actually allows students to separate and visualize D-A# .-A# proteins# and other polypeptides and nucleotide se/uences! ,enes control all life processes! 0herefore# the separation of D-A and gene products provides the potential to examine all of life1s processes! "hat exactly is gel electrophoresis2 &t is a separation technology that is the sum of its parts: gel# a substrate 3thin% of gelatin45 electro# referring to electricity5 and phoresis# from the ,ree% verb phoros# meaning 6to carry across!7 ,el electrophoresis# then# refers to the techni/ue in which molecules are forced across a gel by an electrical current5 activated electrodes at either end of the gel provide the driving force! Arne 0iselius# a +wedish biochemist# won the -obel 8rize for chemistry in '(9* for his wor% with electrophoresis! Exploring the Bar Codes of Life $y using gel electrophoresis# students can gain a uni/ue perspective on D-A structure and function! -ucleic acids# both D-A and .-A# can be separated on the basis of size and charge by means of electrophoresis to identify structural forms of plasmid D-A# as well as determine the size of D-A fragments of genes! :urthermore# the size of un%nown D-A fragments can be determined be constructing a standard curve using the migration distances and sizes of a %nown D-A mar%er! $and patterns of separated D-A on a gel resemble a 6bar code#7 that familiar pattern used to identify consumer products! Each band is a uni/ue 6signature7 revealing a recorded and identifiable D-A fragment! 3+ee below4

+cientists and clinicians regularly use scanning instruments to glean vital information from samples separated by electrophoresis! 0his information is critical in a range of applications: pinpointing cancer types# identifying diseased tissue# characterizing genetic dysfunctions# assessing coronary ris%# and even reading and compiling nucleotide se/uences to map the genomes of many life;forms# including our own! !o" does the techni#ue "ork$ +eparation of large 3macro4 molecules depends on two forces: charge and mass! "hen a biological sample is mixed in a buffer solution and applied to a gel# these two forces act in concert! 0he electrical current from one electrode repels the molecules# while the other electrode attracts them# and the frictional force of the gel material acts as a 6molecular sieve#7 separating the molecules by size and charge!

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-egatively charged molecules will migrate toward the positive pole# while positively charged molecules will migrate toward the negative pole! 0he net negative charge of the phosphate bac%bone results in the D-A fragments having a slightly negative charge and thus will always migrate toward the positive pole! 0he material is roughly analogous to that of a thoroughly wetted sponge# except that in this case# the 6pores7 are submicroscopic! During electrophoresis# macromolecules are forced to migrate through the 6pores#7 away from the closest electrode and toward the farther electrode when electrical current is applied! After staining# the separated macromolecules in each lane can be seen5 they appear as a series of bands spread from one end of the gel to the other! %hat&s in a 'el 0here are two basic types of materials used to ma%e gels: agarose and polyacrylamide! 8olyacrylamide is a material similar to that found in soft contact lenses and is primarily used to separate proteins! Agarose is a natural colloid extracted from seaweed! &n the plant# it helps to resist dessication when exposed to air for extended periods of time Agarose gels have a very large 6pore7 size and are used primarily to separate very large molecules such as D-A with a molecular mass greater than <#*** %dal! =dal is the abbreviation for %ilodalton or '#*** daltons! A Dalton is a unit of molecular weight very nearly e/uivalent to the mass of a hydrogen atom# or '!*** on the atomic mass scale! "hen agarose is heated to about (*> it melts# but solidifies again when cooled below 9)> ! During the solidification process# agarose forms a matrix of microscopic pores! 0he size of these pores depends on the concentration of agarose used! 0ypically the concentration varies from *!)? to <!*?! 0he lower the concentration# the larger the pore size of the gel and the larger the nucleic acid fragments that can be separated! During electrophoresis# D-A molecules wind through the pores in the gel# similar to trying to pass a thread through a stac% of gauze pads! As the pore size decreases 3by increasing agarose concentration4# it is harder for longer D-A fragments to orient properly! +maller D-A fragments can thread their way through the pores# thus migrating faster! (eparation )echni#ues "hile there are other separation techni/ues# the two most prominent in biotechnology are chromatography and gel electrophoresis! $oth processes can simultaneously separate many common molecular entities from molecular mixes! hromatography wor%s best for small molecules 3leaf pigments# for example4# or for large batch;processing re/uirements! Electrophoresis is primarily used as an analytical tool to separate large macromolecules ranging in size from <* to <#*** %dal! 0his techni/ue can separate hundreds# even thousands of macromolecules from one another while using less than a thousandth of a gram of sample material! hromatography typically uses larger samples and can# at best# separate about twenty common molecular groups at one time! AP Biology Lab 6: A Process to *ye +or Pre,Lab Acti-ity 0he five structural formulas 3A;E4 of the stains we1ll be using in the first part of this lab are on the next page! 0he names of each dye and their approximate molecular weights 3@"4 are listed in the cart below! Aoo% carefully at each structural formula and find the charge that each dye molecule possesses! Design a chart in your /uadrilles# and fill in the charge column! *ye .a/es @alachite ,reen Orange , $romophenol $lue rystal Diolet Fylene yanol M% B<( 9)< CC( 9*E )BG Charge

A B C * E

<

Answer the following /uestions# based on the information in the chart: '! 8redict the direction of migration for each of the %nown dye samples 3opposites attract HI;4 <! "hich dye do you predict will move through the gel the fastest2 0he slowest2 B! "rite a brief function for each of the parts used in gel electrophoresis: a! Agarose gel b! Electrophoresis buffer c! "ells in the gel d! Electric current 9! )! Aist one important safety precaution that must be followed when performing any type of gel electrophoresis! Use the data table below as a guide to construct your own table in your /uadrille! @ove on to 6 asting the Agarose ,el7 lab activity when ready!
*ye %ell 0 Migration *istance 1//2 Migration *irection 134,2 *ye Molecules (peed 5ankings

*ye .a/e

Lab Acti-ity Prep: Casting the Agarose 'el

'! <! B! 9! )! C! E! G! (! 8lace the gel casting tray on a flat surface and tape the ends of the trap with mas%ing tape# which will seal the ends of the tray! 0ape usually wor%s better than the end;plates they include with the electrophoresis chambers! &nsert the gel comb# using the G;well side# into the center slots of the tray! 8our approximately <) mA of the melted agarose into the tray# until it reaches a depth of about < mm! .ulers will be at your lab table! Allow the gel to solidify for approximately <*;B* minutes! Do not disturb the gel tray or comb! "hen the agarose has solidified# it will turn opa/ue! After the gel has solidified# carefully remove the comb from the gel! .emove the tape from the ends of the dams! +tore your gels in a Jiploc baggie with a small amount of running buffer# in the refrigerator! "hen you1re ready to proceed: 0he power supply produces a high enough voltage to cause severe electrical shoc% if handled improperly! :or safe operation# follow all directions and precautions! Do not come in personal contact with or allow metal or any conductive material to come in contact with the reservoir buffer or the electrophoretic cell while the power supply is on! Kou should wear protective gear# such as safety goggles and aprons# while loading and running gels!

Part A: A Process to *ye for: 'el Electrophoresis Materials .eeded Per 'roup Dyes to be tested: @alachite ,reen Orange , $romophenol $lue rystal Diolet Fylene yanol Agarose gel 3<!*?4 on gel tray 0$E running buffer 'F# B)* mA @icropipets# '*LA @etric ruler alculator Electrophoresis chamber 8ower +upply Procedure Scenario Kou are a lab technician mixing up a batch of stain 3a mixture of five dyes4 for one of the researchers in the lab! Kou had started ma%ing a large batch when you were interrupted during the procedure! "hen you returned to the stain# you could not remember if you had added all of the components! .ather than waste the whole batch of stain and start over# you will employ agarose electrophoresis to determine if all of the dyes are present in the mixture! Loading and 5unning a 'el You may choose to use the dry, or submarine methods to load your dyes. The following (steps 1-5) describes the dry method. The submarine method follows that. The ry !ethod a! Aane M': @alachite ,reen b! Aane M<: Orange ,

c! d! e! f!
'! <! B!

Aane MB: $romophenol $lue Aane M9: rystal Diolet Aane M): Fylene yanol Aane MC: Un%nown 3mixture4

9!

8lace the tray with the gel on the lab bench! Use a micropipette to load '*LA of each dye sample into the corresponding lane! Do not pierce the bottom of the wells with the micropipette tip! Do not overload wells! 8lace the tray with the loaded gel in the center of the electrophoresis chamber! When filling the electrophoresis chamber with buffer, avoid pouring the solution directly onto the gel and be sure to pour it VERY SLOWLY !f the buffer is poured too "uic#ly, it may wash away the dye samples Add approximately B)* mA of 'F 0$E running buffer to the chamber: +AO"AK pour buffer from a bea%er into one side of the chamber until the buffer is level with the top of the gel! Add buffer to the other side of the chamber until the buffer is level with the top of the gel! ontinue to +AO"AK add buffer until the level is approximately <;B mm above the top of the gel!

The Submarine !ethod (use the same dye arrangement) '! 8lace the gel on the gel tray# in the center of the electrophoresis chamber! <! Add 'F 0$E running buffer to the chamber: +AO"AK pour buffer from a bea%er into one side of the chamber until the buffer is level with the top of the gel! Add buffer to the other side of the chamber until the buffer is level with the top of the gel! ontinue to +AO"AK add buffer until the level is approximately <;B mm above the top of the gel! Loading the gel may be easier to accomplish over a dar# bac#ground, such as the lab table provides $lacing a piece of blac# paper under the electrophoresis chamber will increase the contrast and ma#e the wells easier to see and fill, if no dar# lab table is available B! Use a micropipette to load '*LA of each dye sample into the corresponding lane! Do not pierce the bottom of the wells with the micropipette tip! Do not overload wells! 9! @a%ing sure that the cover# as well as the female jac%s and the plugs# are completely dry# slide the cover onto the electrophoresis chamber! "ipe off any spills on or around the apparatus before proceeding to the next step! )! @a%ing sure that the patch cords attached to the cover are completely dry# connect the red patch cord to the red electrode terminal on the power supply! onnect the blac% patch cord to the blac% electrode on the power supply!

"""#a$e your teacher chec% the connections before proceeding to the ne&t step""". 5unning the 'el:
'! 8lug in the power supply and set it to the desired voltage! !t is recommended that you set the power supply between %&'(&)V *he system will run at a lower voltage but will increase the running time of the gels +*he higher the voltage, the faster the running time, 0urn on the power supply! 0he red power light will illuminate# and bubbles will form along the platinum electrodes in the chamber! Observe the migration of the samples across the gel toward the electrodes! 0urn off the power when the fastest;moving sample has neared the end of the gel! Unplug the power supply! "ait approximately '* seconds# and then disconnect the patch cords from the power supply! .emove the cover from the electrophoresis chamber! arefully remove the gel# on the casting tray# from the electrophoresis chamber 0he dye samples are not fixed on the gel and will continue to diffuse over several hours# ma%ing the bands less distinct! Kou must ta%e measurements on the same day the dye was cast! +%etch your gels# and ta%e measurements on the dye samples! .ecord results in a data table similar to the one in the analysis section!

<! B! 9! )! C! E!

*he dry method has the advantage of ease of loading the gel !t is easy to see the wells when they are not covered with buffer solution *he disadvantage is that one must e-ercise e-treme caution when adding the running buffer to the chamber so as not to wash the samples from the wells *he submarine method has the advantage in that the buffer is already added, and you don.t have to worry about the dyes being washed out of the wells *he disadvantage is that it harder to see the wells underneath the buffer

id you %now' *here are several factors that can affect the speed of migration of a molecule through an agarose gel *he most important are the si/e of the molecule +how physically large it is, and its isoelectric point +the p0 at which the molecule has no net charge, *he concentration of the agarose is also a #ey factor

Analysis 6uestions
'! <! @a%e an accurate and precise s%etch of your gel 3measure# and color4 results in your /uadrille! 3Use appropriate colored pencils# and a metric rulers to do this4 O. ta%e a picture# and tape it in your /uadrille! :or lanes ';)# measure the distance each dye has traveled! @easure the distance from the center of each well to the center of each dye sample! .ecord your results in a table such as the one below! Also note which electrode each dye migrated toward and whether the dye is positively or negatively charged!

Lane ' < B 9 )

Migration *istance 1//2

Migration *irection 13 or ,2

Molecule Charge 13 or ,2

@igration distance will vary based on gel size and the amount of time the gel is allowed to run!
B! 9! )! C! E! "hy did certain dyes migrate toward the positive electrode and others toward the negative electrode2 Examine the dye mixture you placed in lane C! $ased on your results# can you determine whether or not any of the dyes were absent from the mixture2 Now does varying the concentration of agarose used in a gel affect the ability of the gel to separate molecules2 0he gel used in this activity was prepared with <!*? agarose! @any D-A separations are performed on agarose gels varying from *!E? to '!<? agarose! "hat does this tell you about the size of these D-A molecules in comparison to the dyes you used in this lab2 &f you were called away during the electrophoresis procedure and were not able to monitor your electrophoresis run# what do you thin% would happen if the electricity were to remain running in your absence2

Lab 6: Molecular Biology: Part B *.A +ingerprinting Acti-ity: C(7

Background Of the three billion nucleotides in human D-A# more than ((? are identical among all individuals! 0he remaining '? that is different# however# adds up to a significant amount of code variations between individuals# ma%ing each person1s D-A profile as uni/ue as a fingerprint! Due to the very large number of possible variations# no two people 3with the exception of identical twins4 have the same D-A se/uence! :or every '#*** nucleotides inherited# there is one site of variation# or polymorphism! 0hese D-A polymorphisms change the length of the D-A fragments produced by the digestion of restriction enzymes! 0he exact number and size of fragments produced by a specific restriction enzyme digestion varies from person to person! 0he resulting fragments# called 5estriction +rag/ent Length Poly/orphis/s # 3.:A8s4# can be separated# and their size determined# by electrophoresis! @ost of the D-A in a chromosome is not used for the genetic code5 it is uncertain what# if any# use this D-A may have! $ecause these regions are not essential to an organism1s development# it is more li%ely that changes will be found in these nonessential regions! 0hese regions contain nucleotide se/uences that repeat from <* to '** times 3e!g!# ,0 A,0 A,0 A,0 A4# which are the strands that are cut with restriction enzymes to create .:A8s! 0he differences in the fragments can be /uantified to create a 6D-A fingerprint7! Distinct .:A8 patterns can be used to trace the inheritance of chromosomal regions with genetic disorders or to identify the origin of a blood sample in a criminal investigation! +cientists have identified more than B#*** .:A8s in the human genetic code# many of which are highly variable among individuals! &t is this large number of variable yet identifiable factors that allow scientists to identify individuals by the number and size of their various .:A8s! 0his techni/ue is being used more and more fre/uently in legal matters! Using D-A fingerprinting# the identity of a person who has committed a violent crime can be determined from minute /uantities of D-A left at the scene of a crime in the form of blood# semen# hair# or saliva! 0he D-A fingerprint matched to a suspect can be accurate to within one in '* billion people# which is about twice the total population of the world! ertain limitations in the techni/ue prevent two samples from being identified as a 6perfect match7# yet it is possible to measure the statistical probability of two samples coming from the same individual based on the number of %nown .:A8s that exist in a given population! D-A fingerprinting has many other applications! +ince half of the person1s genome comes from each parent# D-A fingerprinting can be used to determine familial relationships! &t has a much higher certainty than a blood test when used to determine fatherhood in a paternity suit! D-A fingerprinting can be used to trac% hereditary diseases passed down family lines and can be used to find the closest possible matches for organ transplants! &t can also be used to ascertain the level of inbreeding of endangered animals# aiding in the development of breeding programs to increase animals1 genetic health and diversity! Kou1ve already experienced the basics of gel electrophoresis# and how you can separate substances using this techni/ue based on charge and mass! 0hrough your reading you have learned about restriction enzymes# and the job they do! &n this part of the lab# you will actually be using a restriction enzyme to cleave a D-A sample! "hen you actually perform a restriction digest# you put the D-A and the enzyme into a small tube and let the enzyme do its wor%! "hile the samples you will be getting have already had that done for you# you will need to centrifuge the contents to re;mix the samples! ,enetic engineering is possible because of these special enzymes that cut D-A! 0hese enzymes are called restriction enzymes# or restriction endonucleases! .estriction enzymes are proteins produced by bacteria to prevent or restrict invasion by foreign D-A! 0hey act as D-A scissors# cutting the foreign D-A into pieces so that it cannot function! .estriction enzymes recognize and cut at specific places along the D-A molecule called restriction sites! Each different restriction enzyme 3and there are hundreds# made by many different

bacteria4 has its own type of site! &n general# a restriction site is a 9 or C base;pair se/uence that is a palindrome! A D-A palindrome is a se/uence in which the 6top7 strand read from )1 to B1 is the same as the 6bottom7 strand read from )1 to B1! :or example: )1,AA00 B1 B1 00AA,)1 0his is a D-A palindrome! 0o verify this# read the se/uence of the top strand and the bottom strand from the )1 end to the B1 end! 0his se/uence is also a restriction site for the restriction enzyme called Eco.&! &ts name comes from the bacterium in which it was discovered! Escherichia coli strain .K 'B 3Eco.4# and 6&7 because it was the first restriction enzyme found in this organism! Eco.& ma%es one cut between the , and A in each of the D-A strands 3see below4! After the cuts are made# the D-A is held together only by the hydrogen bonds between the four bases in the middle! Nydrogen bonds are wea%# and the D-A comes apart!

ut sites: )1,AA00 B1 B1 00AA,)1 ut D-A: )1, AA00 B1 B1 00AA ,)1

0he Eco.& cut sites are not directly across from each other on the D-A molecule! "hen Eco.& cuts a D-A molecule# it therefore leaves single;stranded 6tails7 on the new ends 3see above examples4! 0his type of end has been called a stic%y end because it is easy to rejoin it to complementary stic%y ends! A staggered cut exposes single;stranded regions of the molecule# which are %nown as 6stic%y ends7! -ot all restriction enzymes ma%e stic%y ends5 some cut the two strands of D-A directly across from one another# producing a blunt end! "hen scientists study a D-A molecule# one of the first things they do is to figure out where many restriction sites are! 0hey then create a restriction map# showing the location of cleavage sites for many different enzymes! 0hese maps are used li%e road maps to the D-A molecule! $elow are the restriction sites of several different restriction enzymes# with the cut sites shown!

Eco.&: )1,AA00 B1 B1 00AA,)1

Nind&&&: )1 AA, 00B1 B1 00 ,AA)1

$amN&: )1,,A0 B1 B1 0A,,)1

Alu&:

)1A, 0B1 B10 ,A)1

+ma&:

)1 ,,,B1 B1,,, )1

Nha&:

)1, , B1 B1 , ,)1

"hich enzymes would leave blunt ends2 "hich ones would leave stic%y ends2 *.A +rag/ent Length *eter/ination and (outhern Blotting Under a given set of electrophoretic conditions such as pN# voltage# time# gel type# concentration# etc!# the electrophoretic mobility of a D-A fragment molecule is standard! 0he length of a given D-A fragment can be determined by comparing its electrophoretic mobility on an agarose gel with that of a D-A mar%er sample of %nown length! 0he smaller the D-A fragment# the faster it will move down the gel during electrophoresis! Using a techni/ue called +outhern blotting# the separated fragments are transferred to nitrocellulose paper# labeled with a radioactive probe# and developed against F;ray film! 0he probe# which is coded to bind to specific .:A8s being tested# will develop the film! 0he greater the concentration of D-A in that particular band# the dar%er the band will be! 0he resulting image shows a series of dar% and light bands! 0his pattern is the D-A fingerprint of the tested individual! omparing the distances between the bands in different samples determines the similarities between the samples! .eal human D-A is billions of base pairs long! utting human D-A with restriction enzymes results in millions of fragments on a gel# which is why the +outhern blotting procedure is performed! 0he D-A samples used in this investigation are actually viral D-A# which is much smaller and gives distinct patterns on agarose gels# eliminating the need for +outhern blotting! 0he D-A samples are loaded into wells of an agarose gel and electrophoresed! An electrical field applied across the gel causes the D-A fragments in the samples to move from their origins 3sample wells4 through the gel matrix toward the positive electrode! +maller D-A fragments migrate faster than larger ones# so restriction fragments of differing sizes become concentrated into separate bands during electrophoresis! 0he characteristic number and pattern of bands produced by each restriction enzyme are# in effect# a 6D-A fingerprint7! 0he restriction patterns are made visible by staining with the dye# which binds to D-A! (cenario &nvestigators were called to the scene of a homicide! :ound at the scene was a large amount of blood! $lood typing revealed that not only was the blood of the victim present at the scene# but also blood from another person# assumed to be the guilty party! $lood evidence was collected at the scene! $lood samples from four suspects were also drawn under court order and all samples were found to be of the same blood type as the blood believed to have come from the perpetrator of the crime! 0he police have turned to you and your fellow lab technicians to employ the process of D-A fingerprinting in building a case against one of the suspects Materials .eeded !G? Agarose gel# on gel tray 0$E running buffer 'F# B)* mA +taining tray Aoading Dye @icropipettes @etric ruler Aambda D-AINind&&& mar%er Aab mar%ers @icrocentrifuge tubes Procedures Casting an Agarose 'el D-A stain ,oggles aprons 9 D-A suspect samples Not water bathIincubator Electrophoresis chamber 8ower supply UD transilluminator 8vu && restriction enzyme 8vu && reaction buffer '*F

'! <! B! 9! )! C!

8lace a gel casting tray on a flat surface and tape the ends with mas%ing tape! $e certain that the top edge of the tape aligns with the top of the electrophoresis tray# so that the bottom will be overlapped with more tape! &nsert the gel comb into the gel# using the GOcomb side into the slots near the end of the tray! 8our approximately <) mA melted agarose into the tray# until it reaches a depth of about B mm! @a%e sure you pour slowly to eliminate bubbles# and if bubbles do form# use a pen or pencil to pop them or move them out of the way! Allow the gel to solidify for approximately ');<* minutes! Do not disturb the gel tray or comb! "hen the agarose has solidified it will turn slightly opa/ue! After the gel has solidified# carefully remove the comb from the gel by lifting the comb straight up! .emove the end tape from the tray# and slide your gel out of the tray! 8lace the gel in a Jiploc baggie with about ) mA of 0$E buffer solution and refrigerate over night!

Loading and 5unning a 'el '! 8lace the gel bac% into the tray of the electrophoresis chamber# ma%ing sure the wells are closest to the negative electrode of the chamber! <! Kou will be loading your D-A submarine method! Add approximately B)* mA of 'F 0$E running buffer to the chamber: +lowly pour buffer from a bea%er into one side of the chamber until the buffer is level with the top of the gel! Add buffer to the other side of the chamber until the buffer is level with the top of the gel! ontinue to slowly add buffer until the level is approximately <;B mm above the top of the gel! B! entrifuge the D-A samples in the microcentrifuge to pull the D-A samples to the bottom of the tubes! 9! Aoad '* LA of each D-A sample into the corresponding lane! Do not pierce the bottom of the wells with the micropipette tip! Don not overload the wells! Aane M': D-A mar%er standard Aane M<: rime scene D-A sample Aane MB: +uspect ' D-A sample Aane M9: +uspect < D-A sample )! onnect the lid of your electrophoresis chamber and call your teacher to chec% your apparatus! C! After teacher has chec%ed everything# run your electrophoresis e/uipment for <*;B* minutes 3as time allows in class4! E! Observe the migration of the loading dye down the gel toward the red electrode! 0urn off the power when the loading dye has reached the end of the gel! Unplug the power supply! 0he loading dye is a special dye added to the D-A samples prior to performing electrophoresis and serves two purposes! &t is heavier than the electrophoresis buffer# causing the D-A samples to sin% into the wells! &t is also smaller than most of the D-A fragments in the samples so it runs to the end of the gel faster than the D-A# giving an indication of when to end the electrophoresis run# as you will not see the actual D-A until you stain it! G! "ait approximately '* seconds and then disconnect the patch cords from the power supply! .emove the cover from the electrophoresis chamber! (! arefully remove the gel# on the casting tray# from the electrophoresis chamber! '*! &f time doesn1t allow# you may stop here# by simply placing your gel in a Jiploc baggie with 0$E buffer solution and refrigerating over night again! (taining 'els "ear gloves# apron and eye protection when wor%ing with the /uic% D-A stain! '! ,ently slide the gel from the baggie into the staining tray and pour approximately '** mA of warm dilute stain into the staining tray so that it just covers the gel!

'*

<! B! 9!

)! C!

over the tray and let the gel stain for approximately B*;9* minutesP%eeping a careful and watchful eye on it! @a%e sure the gel remains flat and does not move up against the sides of the staining tray! "hen finished staining# you may decant the stain directly to a sin% drain and flush with water! 0he dilute D-A stain may be saved and reused as well! :or best results# re;warm stain before each use! :lush the gel under streaming tap water 3,E-0AK4! 0hen add distilled or tap water to the staining tray! &t is best not to pour water directly onto the gel! 0o accelerate destaining# gently roc% the tray! Destain until bands are distinct# with little bac%ground color! 0his will ta%e between B* and C* minutes# depending on the amount of agitation and number of water changes! hange the water several times# or destain the gel# without changing the water overnight! Diew the gel against a light bac%ground# such as white paper# or on a light table! ,els can be stored in resealable plastic bags! Once the D-A has been set in the gels# even if the bands fade with time# you may restain the D-A as needed! Accurately s%etch the bands you see on the blan% gel in the Analysis section! $e as exact as possible in s%etching the bands in their actual positions!

Analy8ing your 'el: '! @easure the distance of the D-A bands# in millimeters# from the bottom of the sample well to the bottom of each D-A fragment for the D-A mar%er standard! @easuring to the bottom of each fragment band ensures consistency and accurate measurements! Do not measure the migration distance of the largest fragment nearest to well5 it will not be on the standard curve and will s%ew results! .ecord the measurements in a table of your o"n design! On semi;log graph paper# plot a standard curve for the D-A mar%er standard! 8lot the migration distance in millimeters on the F;axis# against the molecular size in base pairs 3bp4 of each fragment! Draw the best;fit line to your points! When plotting on semi'log graph paper, the fragment si/e on the Y'a-is is e-pressed on a logarithmic scale Label the first series of lines ())

<! B!

+rag/ent *istance Migrated 1//2 Length 1bp2 ; 1top2 < = > ? 6 @ A B ;C bp, 1)) bp, 2oo bp, etc3 *hen label the second series of lines ())) bp, 1))) bp, 2))) bp3etc *he third series would be (),))) bp, 1),))) bp, 2),))) bp3etc 9! @easure the distance that each band traveled for the lanes containing the two suspects and crime scene D-A! .ecord the data in tables of your o"n design 3each one in a separate data table4 )! alculate the base pair size of each of the fragments by moving along the F;axis until you have reached the distance traveled by the fragment! :rom that point# move upward until you intersect the line of best fit on the graph! Determine where that point is on the K;axis and estimate the base pair value at that point! Enter this data in your data tables as well! 3+ee example below4 .emember# you will need four tables li%e this one! One for each of the samples! )ABLE 9.E: *.A Marker (tandard :ie"ing and Photographing 'els

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0ransillumination# in which light passes up through the gel# gives superior viewing of gels stained with D-A stain! A fluorescent light box for viewing slides and negatives provides ideal illumination for D-A stained gels! over the surface of the light box with plastic wrap to %eep the li/uid off the apparatus! A digital camera or cell phone may be used to photograph the gels! apture a photograph of your gel that you can print and put in your lab /uadrille!

Analysis 6uestions '! Examine your stained gel on a light box! ompare the banding patterns formed on each lane of the gel! Now can you further verify whether or not any of the D-A samples tested are the same2 <! "hich of these two suspects do you believe is the real burglar2 Explain your answer! B! Different restriction enzymes are isolated from different types of bacteria and viruses! "hat advantage do you thin% bacteria and viruses gain by having restriction enzymes2 9! &n this lab# you ran your D-A samples on a *!G? agarose gel! "ould you get the same results if you ran your samples on a <? agarose gel2 "hy or why not2 )! 8redict what would happen if you placed your gel in the electrophoresis chamber with the wells containing the D-A samples next to the anode instead of the cathode2 C! Electrophoresis is one method of separating molecules! 8aper chromatography is another method! reate a double bubble thin%ing map showing at least two similarities and two differences between these two methods!

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