GTC LAB 7: Restriction Analysis, Southern Transfer, and Probe Labeling

8/10/2019 GTC LAB 7: Restriction Analysis, Southern Transfer, and Probe Labeling

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GTC Lab 7

GTC LAB 7: Restriction Analysis, Southern Transfer, and

Probe Labeling

In this lab you will analyze the restriction digests of your ATT recombinant clones from

the previous laboratory period by agarose gel electrophoresis. As a second part of the

analysis you will perform !outhern hybridization "a !outhern #blot$% on the resolvedfragments to detect a shift in size of the appropriate vector band which will indicate that

the gene was cloned into the correct position.

Southern hybridization

!outhern transfer and hybridization was a techni&ue developed in '(7) by

*dward !outhern. The techni&ue has several applications but primarily is used to studyhow genes are organized within chromosomal +,A. It may also be used to analyze

cloned se&uences. -ou will be using this techni&ue to determine whether you have

cloned the gene into the correct site.

In general your plasmid +,A is first digested with one or more restriction enzymes andthe resulting fragments are resolved by agarose gel electrophoresis. The +,A in the gel

is then denatured and transferred from the gel to a solid support "a nylon or nitrocellulosemembrane%. The +,A binds strongly to the membrane and is thus immobilized in the

eact positions that it was following gel electrophoresis. The +,A attached to the

membrane is hybridized to a labeled nucleic acid probe "+,A or /,A% and the bandscomplementary to the probe are identified by procedures that will detect the label on the

probe "for eample fluorescence detection if the label is a fluorescent molecule%.

Southern blot stes:

'. !eparate +,A by agarose gel electrophoresis.0. +enature +,A in gel and blot "transfer% +,A fragments to a membrane.1. 2ybridize immobilized ss+,A with a single3stranded probe complementary to the

se&uence of interest.

The probe must be #labeled$ with a mar4er for detection.

5. 6ash off ecess unhybridized probe.

). +etect "visualize% the probe "where it has hybridized%.

Procedure:

!" Run your digestsof ATT recombinants from the previous lab on a .89 agarose gel.

:a4e up ) mL of agarose gel solution with .) ;L *tL of loading buffer ? /,ase to each digest load 0 ;L of each and run

the gel at '@ for 5) minutes.

Bhotograph the gel. Include a ruler in the picture to measure the distance from the

wells to the bands. This will be used later to interpret the results of your !outhern

blot. Cut off one corner of the gel for orientation.

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!!" Southern Transfer

-ou will be transferring your +,A under al4aline conditions to a nylon membrane. This

will result in covalent crosslin4ing of the +,A to the positively charged nylon.

'. Transfer the gel to a small plastic tray. sing a razor blade cut off the top portion of

the gel from immediately below the wells. Cut one corner of the gel for orientation.

0. !oa4 the gel for ') minutes at room temperature in several volumes "this means

several volumes of your total gel volume% of al4aline transfer buffer. This step

denatures the +,A in the gel.

#ear glo$es %hen handling the denaturing solution.

1. Change the solution and continue to soa4 the gel for another ') minutes "minimum%.

5. Cut a piece of nylon membrane eactly the size of the gel. 6ear gloves to prevent

transferring greasy fingerprints to the membrane. Cut a corner of the membrane that corresponds to the cut corner of your gel. Dloat the membrane on the surface of some

deionized water in a plastic weighboat until it wets completely. -ou will be able to see

a change in the membrane color from white to more translucent.

). 6hile the +,A is denaturing set up your transfer apparatus. Blace a piece of blotting

paper on a glass plate supported on the edges of a plastic transfer dish. This %ill be

de&onstrated in class"The ends of the blotting paper should drape over the edges of the plate. Dill the dish with transfer buffer until the level of the li&uid reaches almost to

the top of the support "glass plate%. 6hen the blotting paper on the top of the support is wetted roll a glass pipet over the surface to eliminate the air bubbles.

E. Ta4e the gel from the solution in step 1 flip it upside down so that the underside is

now on top and center it on top of the blotting paper on the support. /un a gloved finger or roll a glass pipet over the surface to ensure that there are not bubbles trapped

between the gel and the support.

7. !urround the gel with strips of parafilm.

8. Blace the wet membrane on top of the gel. Chase out any bubbles between the gel and

the membrane using a gloved finger or by rolling a glass tube over the top.

(. Cut two pieces of blotting paper the eact size of the gel. 6et them in transfer buffer

then place them on top of the wet membrane.

'. Cut a stac4 of paper towels the eact size of the gel. The stac4 should be at least E cm

high. Blace the stac4 on top of the blotting paper.

''. But a glass plate on top of the stac4 and weigh it down with a small flas4.

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GTC Lab 7

'0. Allow the transfer to proceed by capillary action overnight.

The ne't day:

'1. +isassemble the transfer #sandwich$ and soa4 the membrane in neutralization buffer for ') minutes at room temperature. This removes any pieces of agarose stic4ing to

the membrane and neutralizes the membrane.

'5. Blace the membrane on a paper towel to dry. :a4e sure the +,A side "the side

facing down on the gel% is B. It will be retrieved from your bench and stored by a

lab instructor at the end of the day.

Part !!!: Probe labeling

The probe for the !outhern blot will be labeled with digoigenin "+IG% which is a steroid

hapten. +IG is conFugated to dTB which will be incorporated into your probe by polymerase

activity. +IG3dTB incorporated into the probe is then detected by an antibody "anti3

digoigenin% that is covalently bound to the enzyme al4aline phosphatase. The al4alinephosphatase substrate C!B+ is then put on the membrane. *nzymatic dephosphorylation of

C!B+ by al4aline phosphatase leads to a light emission at a maimum of 577 nm which isdetected by eposure to 3ray film for 1 minutes. Thus the light will be emitted only where

the antibody has bound to the +IG.

(a)ing the robe * o$er$ie%:

+IG3dTB will be incorporated into the probe by the random prime method. In this techni&ue

the probe +,A to be labeled is first denatured into single strands. Then random heamers "E3

nucleotide single3stranded primers in all random combinations of nucleotide se&uence% areannealed to the single3strand probe template. d,TBs "including the labeled d,TB in this case

+IG3dTB are added along with Hlenow enzyme in a reaction buffer and the primer is etended

by polymerase activity which also incorporates the labeled nucleotide into the growing chain.

Procedure:

'. -ou will start with ' ng of +,A label. The concentration of the stoc4 will be provided

in class. Add sterile water to a final volume of 'E >L in a microcentrifuge tube.

0. +enature the +,A by heating in a 'C heat bloc4 for ' min and &uic4ly chilling in an

ice bath.

+ote: Co&lete denaturation is essential for efficient labeling"

1. Add 5 >L of the labeling mi to the denatured +,A mi and centrifuge briefly.

5. Incubate the reaction overnight in a 17C waterbath.

). -our lab instructor will stop the reaction by heating the reaction to E)C for ' min. This denatures and inactivates the Hlenow enzyme. -our probe will be stored

at 30C until you are ready to probe your blot "net wee4%.

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GTC Lab 7

Co&letion of Southern blot: hybridization, %ashing, and de$eloing

'. Insert your dry blot into a hybridization tube +,A side facing inward. Add 7 mL

of hybridization solution to the tube. !eal the tube and incubate for 1 minutes in the

hybridization oven. This is the #prehybridization$ step. It prevents nonspecific

binding of the probe to the membrane.

0. +enature the +IG3labeled +,A probe in the heat bloc4 "()3'C% for ) minutes.

/apidly cool in an ice bath.

1. Add 0 ;L of the denatured probe to ' mL of hybridization buffer mi briefly and add

the entire mi to the hybridization tube. Incubate in the hybridization oven at 50C overnight.

5. The net day wash your blot 0 ) min in ' mL Blot #ash "0 !!C .'9 !+!% at room temperature with periodic gentle agitation by hand.

). 6ash 0 ') min in ' mL preheated Blot #ash -".) !!C .'9 !+!% at E8C.

E. /inse the membrane for ' minute in 0 mL #ashing buffer.

7. Incubate for 0 min in ' mLBloc)ingsolution.

8. Incubate for 1 min in 0 mL Antibody solution.

(. 6ash 0 ') min in ' mL #ashing buffer.

'. *&uilibrate 0 min in 0 mL .etection buffer.

''. Blace membrane with +,A side facing up on a piece of !aran wrap. Apply 8 >L

of C!B+ substrate "from the brown bottle%. !pread evenly over the membrane surface. Incubate for ) min at room temperature.

Rock back and forth periodically to ensure even coating across the surface of the

membrane.

'0. +rain ecess substrate solution from the surface of the blot. Blace on a fresh piece

of !aran wrap. Cover the top with !aran.

'1. *pose to 3ray film for ) min at room temperature.

'5. +evelop the film in the dar4room as shown by an instructor.

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GTC Lab 7

GTC LAB 7 / !+STR0CT1R2S G0!.3

Prere4uisite infor&ation:!ome prior discussion about !outhern blotting techni&ue.

Students %ill gain:

Bractice applying !outhern blotting techni&ue

Ti&e: Approimately 5 hours

(aterials:

microcentrifuge tubes

microcentrifuge

denaturation solution ".5 , ,aJ2% 0 mLs per gel to transfer

neutralization buffer

17K water bath

ice and ice buc4ets "one per pair%

6hatman paper or thic4 blotting paper

small plastic or glass dishes for gel denaturation and transfer

agarose

@ visible ruler "for photographing gel%

' 4b ladder

' sample loading buffer with /,ase

' TA* running buffer

nylon transfer membrane

large weighboats or other containers to moisten nylon membrane

paper towels

razor blades "or small scissors to cut corner of membrane%

+,A for probe "' ng should be enough for '3'0 blots%

heat bloc4

hybridization oven

+IG random prime labeling 4it "Amersham%


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GTC Lab 7

Al)aline transfer buffer: .5 , ,aJ2= ' : ,aCl

+eutralization buffer: .) : Tris3Cl "p2 7.0%5 ' : ,aCl

-6' SSCM 1 : ,aCl .1 : sodium citrate adFust p2 to 7.

#ashing bufferM :aleic acid sodium chloride Tween 0 "detergent% as per

manufacturers protocol

Bloc)ing solutionM )9 dried mil4 dissolved in maleic acid buffer "manufacturers

protocol%

Antibody solutionM 'M' dilution of anti3+ig antibody in bloc4ing solution

.etection bufferM Tris and sodium chloride buffer solution at p2 (.) "manufacturers

protocol%

General:

The !outhern blot is an etra eercise N it is not re&uired to identify correct ATT

recombinants. Correct recombinants can be identified according to their bandingpattern on the gel following


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GTC Lab 7

*pected resultsM The '.1 4b band should be hybridized on the lane of vector

alone digested with

GTC LAB 7: Restriction Analysis, Southern Transfer, and Probe Labeling

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