Southern Blotting

MSU Potato Lab

A. DNA AGAROSE GEL

1. Prepare a 1% agarose gel and load 5 μl of BMB Molecular Weight Marker DIG
labeled in one lane.

2. Mix 20μg Plant Genome DNA (which has been digested with an enzyme or
enzymes that will produce a fragment of DNA, encoding the desired gene, of a size
between 1 and 8 kb, if possible but larger or smaller fragments should still work)
with Running Dye (1X final). Centrifuge briefly and load on the gel. Be sure that a
negative control (non-transgenic plant DNA digested the same) is included on the
gel. As a positive control a small amount (~10ng) of the plasmid can be digested
to cut out the gene (used for making the probe) and run on the gel. Since this will
produce a bright signal, run this sample in the last lane; preferably separate it from
a plant lane with a blank lane. With large gels, 15 cm, it is best to run overnight at
about 15-20 volts to ensure sharp band separation.

3. When finished, photograph the gel with a fluorescent ruler. Mark the upper left
hand corner of the gel by cutting off the corner with a spatula.

4. Place the gel into a plastic tray and DEPURINATE by incubating 10 min with
0.25N HCl (250 ml or enough to cover gel completely). Agitate gently. AVOID
LONG INCUBATIONS. This process cleaves DNA that allows large fragments to
transfer to the nylon membrane.

5. Wash briefly twice with about 250 ml ddH20.

6. DENATURE with 250 ml of SOLUTION D, 15 min. Agitate gently.

7. Repeat.

8. Wash briefly twice with 250 ml ddH2O.

9. NEUTRALIZE with 250 ml SOLUTION N, 15 min. Agitate gently.

10. Repeat. The gel can be left in this solution for a short time until ready to be
installed onto the blotting stack.

B. INSTALLATION OF THE BLOTTING STACK

1. Wrap a plastic platform, the size or larger than the gel, with Whatman paper and
SOUTHERNS, page 2

place in a plastic container. Fill container 1/3 full with 20x SSC. Allow
the Whatman paper to become saturated with the solution.

2. Saturate 3 pieces of Whatman paper a little larger than the size of gel with 20x
SSC. Place on top of the platform. Remove air bubbles by rolling a clean test tube
over the top.

3. Place gel upside down onto the Whatman paper and remove any trapped air
bubbles under the gel by pressing with fingers of your gloved hand. (Gel is placed
upside down because the front gel wells are ridged. A flat transfer surface is on the
back of the gel.)

4. Cover the gel with a large piece of plastic wrap and with a razor blade cut the
plastic wrap around the edge of the gel. Remove the plastic that is on the gel
leaving a frame around sides. This ensures that the capillary movement of buffer
will only go through the gel.

5. Cut a piece of nylon membrane (Amersham Hybond N) the size of the gel. DO
NOT TOUCH THE MEMBRANE WITH YOUR FINGERS! This can cause high
background. Use blunt ended forceps and only touch corners!! Wet the nylon
membrane with 20x SSC. Carefully lay it on top of the gel, if an air bubble is
present lift up on that part of the membrane and lay it down again. Do not touch
the membrane or roll a test tube directly on it to remove air bubbles.

6. Saturate 2 pieces of Whatman paper with 20x SSC and place on top of the nylon
membrane. Make sure there are no air bubbles by rolling a clean test tube over the
top.

7. Cut a stack of paper towels ∼2 inches thick to fit into the plastic tray and place dry
on top of the Whatman paper.

8. Place a flat piece of plastic on top of the blotting stack. Place on top of that a 500g
- 1kg weights (e.g. a flask or bottle with water).

9. Allow transfer to proceed overnight.

C. TAKING DOWN BLOT

1. With gloved hands remove the layers of the blotting stack down to the nylon
membrane. With a pencil, mark the positions of the gel wells on the nylon
membrane.
2. Mark the upper left corner of gel by cutting the corner of the nylon membrane off .
SOUTHERN,page 3

3 Place the membrane into a tray with 5x SSC for 5 min. Agitate gently. DNA side
up.

4. Place the membrane DNA side up onto paper towels.

5. To Bind DNA to the membrane, EITHER place the blot between two pieces of
Whatman paper and bake at 80° C in an oven for 2 hours or use the Stratalinker on
the Autocrosslink mode on the DNA side and 200 Joules on the other side.

6. Store the dry blot between sheets of filter paper and in a plastic bag at room temp.
or for long storage >1 week store at 4°C until ready for the hybridization
experiment.

Solutions:

1) 0.25N HCl Stock HCl is 12N 20.8ml of HCl

979.2ml of ddH2O
= 1 Liter

2) Solution D Denature solution 1.5M NaCl 87.75g

0.5M NaOH 20.00g
ddH20 to 1 Liter

3) Solution N Neutralizing solution 0.5M Tris 60.50g

1.5M NaCl 87.75g
1mM EDTA 0.372g
ddH2O to 1 Liter
pH to 7.5

4) 20 X SSC 3.0M NaCl 175.30g

0.3M NaCitrate 88.2g
ddH20 to 1 Liter
pH to 7.0
 SOUTHERN HYBRIDIZATION
USING BOEHRINGER MANNHEIM NONRADIOACTIVE
NUCLEIC ACID DETECTION SYSTEM

A. PREHYBRIDIZATION

1. Place the DNA southern blot into a small hybridization tube by carefully rolling it with
the DNA side toward the inside of the tube. Use gloved hands and blunt ended forceps.

2. Warm 20mls of prehybridization solution to 420C.

3. Boil 500ul of Salmon Testes DNA (10mg/ml) for 10min and then place on ice for 2
min. Add the 500ul of DNA to the 20mls of prehybridization solution. The final
concentration of DNA is 250ng/ml. This is used to block nonspecific binding sites on the
blot.

4. Add the prehybridization solution to the blot in the tube and place tube in
hybridization oven that has been warmed to 420C. MAKE SURE YOU BALANCE THE
TUBE WITH ANOTHER TUBE OF EQUAL WEIGHT!

5. Prehybridize blot for at least 2hr at 42oC. Longer times are possible.

B. HYBRIDIZATION

1. Prepare hybridization solution for hybridization as follows. Hybridize a 15x15cm blot

with at least 6mls of solution and no more than 20mls. The amount varies depending on
the amount of stock probe that you have. Warm the Hybridization solution to 420C.

2. Denature Salmon Testes DNA by boiling. (The final concentration of S.T.DNA should
be 125 ng/ml in the hybridization solution.) After boiling place on ice 2 min.

3. Denature the DIG-labeled probe DNA by boiling for 10min. (The final concentration
of the probe should be 10ng/ml). After boiling, place tube on ice for 2min.

4. Add the probe and Salmon DNA to the warm hybridization solution and mix by
shaking well.

5. Remove prehybridization solution from hybridization tube. (The prehybridization

Page 2

solution can be reused 2 times. Store at -200C. To reuse, the Salmon DNA in the solution
must be denatured. Place the solution in a 650C water bath for 15min. The flashpoint of
pure formamide is 680C therefore do not boil the solution.)

6. Do not let blot dry in tube. Immediately add the hybridization solution and place in
hybridization oven. Make sure the tube is balanced! Incubate overnight at 37oC.

C. STRINGENCY WASHES

1. Remove tubes from oven and increase the oven temperature to 650C for future use.
Remove hybridization solution. Do not let blot dry in tube. (The hybridization solution
can be saved. Store in a 15 or 50ml tube labeled very clearly with the name of probe and
# of times used. Reuse 1 or 2 times. To reuse thaw and denature by heating the solution at
650C for 10 min. Cool to hybridizing temperature and add to blot.)

2. Immediately wash membrane twice in the hybridization tube with about 50mls of 2X
Wash solution for 15min per wash at room temp.

3. Warm 0.5X Wash solution in 650C water bath and add 50ml to membrane in
hybridization tube. Place in hyb. oven which has been warmed to 650C and incubate for
15min. Remove solution and add fresh warmed solution and incubate another 15 min in
the oven.

4. Proceed directly to the detection procedure.

NOTE: HYBRIDIZATION AND STRINGENCY CONDITIONS:

DIG-Labeled probes demonstrate the same hybridization kinetics as radiolabeled probes.

Labeled probes can hybridize non-specifically to sequences that bear homology but are
not homologous to the probe sequence. These hybrids are less stable than perfectly
matched hybrids. They can be dissociated by performing washes of various stringencies.
The stringency of washes can be manipulated by varying the salt condition and
temperature. It is recommended that one should hybridize stringently, i.e. optimize
hybridization conditions (increase temp., but not higher than 550C) rather than washing
stringently.
Page 3

D. SOLUTIONS

1. Prehybridization and Hybridization solution:

Concentration Amount of Stock solution

5X SSC 75ml of 20XSSC

2% Block solution 60ml of 10% Block Solution
0.1% N-lauroylsarcosine 3ml of 10% N-lauroylsarcosine
0.2% SDS 6ml of 10% SDS
ddH2O 6ml of ddH2O
Formamide (deionized)* 150ml of Pure Formamide
300ml Total

Aliquot in 50ml tubes and store at -200C until use.

Salmon Testes DNA and Probes are added just before use.

*Deionizing Formamide: Just before preparing the above solution place the desired
amount of formamide into a beaker in the fume hood. Add mix resin beads 15g/150ml of
formamide to the beaker. Mix with stir bar for 1 hour.
Filter the formamide through a piece of Whatman paper. Now it is ready to add to the
Pre/Hybridization solution.

2. 2X Wash Solution:

Concentration Amount of Stock solution

2X SSC 100ml of 20X SSC

0.1% SDS 10ml of 10% SDS
ddH20 890ml of ddH20____
1 Liter

3) 0.5X Wash Solution:

Concentration Amount of Stock solution

0.5X SSC 25ml of 20X SSC

0.1% SDS 10ml of 10% SDS
ddH20 965ml of ddH20
Page 4

E. DETECTION

1. After hybridization and stringency washes, rinse membrane briefly with WASHING
BUFFER about 2-5 min in a plastic tray with the DNA side up.

2. Incubate membrane in 80mls of BUFFER 2 for 30 min.

3. Dilute anti-DIG-AP conjugate 75 mU/ml (1:10,000 dilution) =2ul in 20ml of fresh

BUFFER 2.

4. Incubate membrane for 30 min in the antibody solution on shaker either in a plastic
tray or in a zip lock bag. Ensure that the solution is covering the entire blot with gentle
agitation.

5. In a plastic tray wash the membrane 2X 15 min. with 100ml of WASHING BUFFER.
Apply gentle agitation.

6. Equilibrate membrane 2-5 min. in 20 ml BUFFER 3.

7. Dilute CSPD (25mM) a 1:100 dilution in BUFFER 3. A 15X15ml blot will need 2ml
of this solution. Add 20ul of CSPD to 1.8ml of Buffer 3. (Note the diluted CSPD can be
reused 1X. After use collect the 2mls and place in a 15ml tube, wrap with foil and label.
Place at 40C.)

8. Place blot on a piece of Whatman paper to remove excess liquid. DO NOT LET DRY.
Using forceps lift and place in a clear plastic sealable folder DNA side up. Pipet the
diluted CSPD solution onto the blot. Carefully close the folder. Ensure that the entire blot
is covered with the solution. Once closed, immediately seal the folder closed with the
sealer. Incubate blot in dark for 5 min.

9. Cut open the folder. With forceps remove blot and place on a piece of Whatman paper
to remove excess moisture. DO NOT LET DRY. Keep blot DNA side up.

10. Place the membrane in a new plastic folder and seal closed with the sealer. Incubate
for 10 min at 370C. (This enhances the luminescent reaction.)

11. Expose the blot to film. (CSPD is a chemiluminescent substrate for alkaline
phosphatase that enables extremely sensitive and fast detection of biomolecules by
producing visible light that can be recorded on film. A delay in reaching maximum light
emission is observed however the signal persists for several days on the blot.) I usually
incubate the blot for 4-6 hours before the initial exposure to film. An exposure of 15min
to 1hour should be sufficient however you might have to adjust according to the intensity
of signal
Page 5

on your blot. CSPD reaches a peak of emission after about 12 hours. Store blot at room
temp. in the dark between exposures. Once exposures are completed store blot at room
temp. labeled. Blots can be stripped and reprobed.

SOLUTIONS:

1) Maleic Acid Buffer

Concentration Amount of Stock solution

0.1M Maleic acid 11.60g
0.15M NaCl 8.77g______________
pH to 7.5 using NaOH pellets
add slowly until pH is reached
then add the rest of ddH20 to 1 liter.

2) WASHING BUFFER

Concentration Amount of Stock solution

Maleic Acid Buffer Make the same as above

0.3%Tween 20 3ml for 1 Liter

3) BLOCKING STOCK SOLUTION 10X conc.

Blocking reagent from BMB, 10% (w/v) in maleic acid buffer. Dissolve blocking
reagent by constantly stirring on a heating block at (650C). Do not boil the solution. It is
difficult to get into solution and may take several hours. Be sure that all of it has
dissolved and then autoclave. Store at 40C. The solution is opaque.

A much cheaper alternative to BMB blocking reagent is non-fat dry milk. To prepare
dissolve 10% (w/v) in maleic acid buffer. DO NOT autoclave this solution. Aliquot and
store at -200C. Short term storage at 40C ( 2-3days).

4) BUFFER 2 (make fresh for each use)

Concentration Amount of Stock solution

1% Blocking Buffer 10ml

Maleic Acid Buffer 90ml
Page 6
5) BUFFER 3

Concentration Amount of Stock solution

0.1M Tris-HCl 12.1g

0.1M NaCl 5.84g
ddH2O 600ml
50mM MgCl2 25ml of 2M stock_(only needed for color detection not
CSPD detection) pH to 9.5
add ddH2O to 1 Liter

6) STRIPPING BUFFER

Concentration Amount of Stock solution

0.1%SDS 10ml of 10% SDS stock

ddH2O 990ml_______________
1 Liter

7) Anti-digoxigenin (DIG)-AP(alkaline phosphatase conjugate) Fab fragments:

Boehringer Mannheim Cat.No. 1093 274, 150 Units

8) CSPD Chemiluninescent Substrate:

Boehringer Mannheim Cat.No. 1655 884, 1ml (25mM)
 NON-RADIOACTIVE DNA PROBE ISOLATION
PURIFICATION AND RANDOM PRIMED LABELING
USING BOEHRINGER MANNHEIM DIGOXIGENIN SYSTEM

A. PROBE SELECTION AND ISOLATION

Part 1: Probe Selection and Template Preparation

1. Selection of a probe: The probe should consist of the gene of interest. If possible it
should not include any portion of the vector as well as bases outside of the recombination
sites. The probe should be greater than 200bp and less than 10kb.

2. Obtaining template DNA: For Random Primed labeling it is most efficient to start
with 3ug of template DNA, this should provide enough labeled probe to hybridize > 15
blots or more. The labeling can be scaled up if larger amounts are desired. The
following are 2 procedures that can be used to obtain the template DNA.

a) If the gene of interest is in a plasmid in which you have PCR primers for and can
amplify the gene of interest (e.g. a Bluescript plasmid see below) then amplify 4ug or
more (this will give you some to spare) of the gene using the PCR protocols described for
that plasmid and primers. After the PCR, test 10ul of reaction on an agarose gel to check
for amplification and then quantify your DNA. Proceed to Part 3.

Procedure for PCR amplification of genes cloned into Stratagene’s Bluescript

plasmid called pBS (SK+):

1a. Currently Cry5, PVY and NPTII genes have been cloned into the polylinker site of
the pBS (SK+) plasmid vector and the strains are stored in the -80o freezer. Streak the
strain of choice on a LB agar plate containing Ampicillin antibiotic at a concentration of
100ug/ml. Incubate plate at 37oC O/N.

2a. Inoculate 3mls of LB containing 100ug/ml of Ampicillin with an isolated colony from
the O/N plate. Incubate O/N at 37oC on shaker.

3a. Isolate the plasmid DNA from 1.5ml of the culture using the standard Alkaline Lysis
Plasmid Isolation procedure which can be found in “Molecular Cloning” by Sambrook
and Maniatis or by using Promega’s Wizard Miniprep Plasmid Isolation System
following the instructions given in the kit. When complete quantitate the isolated
plasmid.
page 2

4a. Setting up the PCR reaction. Two reactions of 100ul volume should produce an
amount of DNA in excess of what will be needed for the template in the probe synthesis
procedure.

In each tube add: 100ul

Taq Polymerase Buffer 10X 10ul

dNTP mix 10mM 2ul
MgCl2 50mM 3ul
Plasmid DNA 100ng
T3/T7 primer set from Stratagene 4ul
Taq polymerase 0.5ul
ddH2O to 100ul final volume

Centrifuge for 5sec to bring solution to bottom of tube.

Overlay solution with 30 ul of sterile mineral oil.

5a. Start PCR cycle:

94oC 5min

then 30 cycles of:

94oC 1min
o
52 C 1min
72oC 2min

after cycles:
72oC 5min
4oC Holding temp.

6a) Check 10ul of the PCR reaction on an agarose gel to confirm size and quality. If it
looks good then determine the concentration. Proceed to Part 3.

b) If the gene of interest is not in a plasmid with PCR primers, than you will need to
purify at least 100ug of plasmid DNA in order to obtain >3ug of the template DNA. This
can be done by using a QIAGEN Midi Plasmid Purification Kit.

Qiagen Midi Plasmid Protocol

1b. Growth of bacterial cultures: grow the bacteria in the presence of the selective
antibiotic. The quantity of culture will depend on the copy number of the plasmid. Grow
enough culture to obtain 100ug of plasmid. Bacterial cultures should always be grown
from a single colony grown on a selective plate.
page 3

2b. Centrifuge the O/N culture at 5000rpm for 10min. Remove supernatant.

3b. Add RNase A to a concentration of 100ug/ml to the Buffer P1. Add 4ml of this to the
bacterial pellet. The bacteria should be resuspended completely, leaving no clumps.

4b. Add 4ml of Buffer P2, mix gently, and incubate at room temperature for 5 min. and
not longer. DO NOT vortex, mix by inversion. Vortexing shears the genomic DNA and it
will contaminate your sample. Close buffer P2 immediately after use to avoid the
reaction of the NaOH with CO2 in the air.

5b. Prechill Buffer P3 on ice. Add 4ml of P3 and mix gently by inversion immediately.
A white precipitate will form. Incubate 15 min on ice.

6b. Centrifuge at > 20,000 x g for 30min at 40C. Remove the supernatant immediately
and save. Discard pellet.

7b. Equilibrate a Qiagen-tip 100 by applying 4ml of Buffer QBT and allow the column to
empty by gravity flow. The column will not dry out. Do not force out the remaining
buffer.

8b. Apply the supernatant to the column and allow it to enter the resin by gravity flow.
Once the column is empty, wash the column by applying 2x 10ml of Buffer QC. Allow it
to move through the column by gravity flow. Do not force out traces of buffer, it will not
effect the elution.

9b. Elute the DNA with 5ml of Buffer QF, allowing it to flow through by gravity.

10b. Precipitate the DNA with 0.7volumes of room temperature isopropanol. Centrifuge
immediately at 15,000 x g for 30 min. at 40C. Carefully remove the supernatant. pellets
from 2-propanol precipitation have a glassy appearance and may be more difficult to see
than the fluffy salt containing pellets with ethanol, therefore mark the outside of the tube
before you centrifuge so you can avoid the pellet.

11b. Wash the pellet with 70% ethanol room temperature and redissolve pellet in ddH2O.
Determine the amount of DNA purified with a spectrophotometer. Proceed to Part 2.
Page 4

Solutions For Qiagen:

QIAGEN Plasmid Midi Kit (25): includes tip-100 Reagents and Buffers, Qiagen Inc.
9600 De Soto Ave. Chatsworth, CA 91311 Cat. No. 12143.

Provided in Kit:

Buffer P1 Buffer QBT Tip-100 column

Buffer P2 Buffer QC RNase A
Buffer P3 Buffer QF
Note: See Qiagen Handbook for recipes for the above solutions if they run out.

Part 2: Digestion of Plasmid.

1. Once the plasmid has been purified, proceed with the restriction digestion of the
plasmid to produce a fragment of DNA that can be used as a probe.

2. Run a test digest with the enzymes selected on the plasmid and run on an agarose gel
to ensure that the correct size fragment appears.

3. Once you have determined which enzymes you will use, digest enough of the plasmid
so that you will have at least 4ug of the DNA fragment itself.

Part 3: Isolation of Probe DNA Using Qiagen Qiaquick Gel Extraction Kit.
Qiagen Qiaquick Gel Extraction Kit:

Follow instruction given in kit.

B: Random Primed DNA-labeling with Digoxigenin-dUTP.

1. The reagents are available separately or in the GENIUS DIG DNA Labeling Kit
from Boeringer Mannheim Cat.No. 1175 033. See solutions section for individual
Cat.No.

2. The DNA sample should be 500ng-3ug. The 3ug of DNA is most efficient. The sample
should be in ddH2O at a volume of 15ul as described above in part 3.

3. Heat denature the DNA template in a boiling water bath for 10 minutes, and quickly
chill it on ice for 30 seconds before use. Briefly centrifuge tube 10 sec.

4. Add 2ul Hexanucleotide mixture (10X) and 2ul dNTP labeling mixture (10X) to the
tube on ice.
Page 5

5. Add 1ul of Klenow enzyme, labeling grade, to the tube for a final concentration of
100U/ml, and mix using the pipet tip. Incubate the reaction tube at 370C O/N (20Hrs) to
obtain 890ng of synthesized DIG labeled DNA. Shorter times will result in less labeled
probe.

6. After incubation add 2ul of 0.2M EDTA pH 8.0 to stop the reaction.

7. For all labeling reactions it is extremely important that you verify the labeling
efficiency in a direct assay prior to hybridization. Proceed to section C.

Solutions for Random Primed DIG-labeling Procedure:

DIG DNA Labeling Kit, Random Primed DNA-labeling, from Boeringer Mannheim, Cat.
No. 1175 033

Hexanucleotide Mix, Cat.No. 1 277 081

dNTP Labeling Mix, Cat.No. 1277 065
Klenow Enzyme labeling grade, 100 units, Cat.No. 1 008 404

Additionally required solutions:

0.2M EDTA pH 8.0

Page 6

C: Estimating the Yield of DIG-labeled Probes.

1. Make serial dilutions of the DIG-Labeled control in DNA Dilution buffer according to
the following dilution scheme:

Labeled
Control Stepwise Final Total
DNA Dilution Conc.(tube) Dilution

5ng/ul 2ul in 8ul buffer 1ng/ul(A) 1:5

1ng/ul 2ul in 18ul buffer 100pg/ul(B) 1:50

100pg/ul 2ul in 18ul buffer 10pg/ul(C) 1:500

10pg/ul 2ul in 18ul buffer 1pg/ul(D) 1:5000

1pg/ul 2ul in 18ul buffer 0.1pg/ul(E) 1:50000

A->E are the Control dilutions which will be used as standards to quantify your labeling
reaction. These samples can be stored at -200C for continual use.

2. Dilute your probe labeling reaction by making a serial ten-fold dilution.

Dilution series for probe labeling reaction:

Amount of
Dilution
Tube Buffer Total Dilution

Probe 2ul in 18ul buffer 1:10(A)

(A) 2ul in 18ul buffer 1:100(B)

(B) 2ul in 18ul buffer 1:1000(C)

(C) 2ul in 18ul buffer 1:10000(D)

(D) 2ul in 18ul buffer 1:100000(E)

Page 7

3. Spot 1ul of each of the dilutions made in step 1 and 2 onto a small piece of nylon
membrane, marking the membrane with a pencil to identify each dilution. Mix the
dilutions very well just before spotting on the membrane.

4. Fix the nucleic acids to the membrane by crosslinking with the Stratalinker set at
autocrosslink.

5. Wet the membrane with 50ml of Washing Buffer.

6. Incubate the membrane in Buffer #2 for 5min at room temperature on shaker with
gentle agitation.

7. Dilute anti-DIG-alkaline phosphatase antibody 1:5000 in Blocking Solution 10ml.

Place membrane in a small zip lock bag with the 10mls of antibody solution. Incubate the
membrane for 10 min. on shaker with gentle agitation. The diluted antibody must cover
the entire blot.

8. Remove membrane from bag and place in a plastic tray. Wash the membrane 2X 5min
in Washing Buffer at room temp. While this is incubating mix 45ul of NBT solution and
35ul X-phosphate solution in 10ml of Detection Buffer. This freshly prepared color
substrate solution should be protected from light until use.

9. Incubate the membrane in Detection Buffer #3(with the MgCl2 for 2 min. Now place
the membrane in a zip lock bag and add the diluted color substrate solution to the bag,
seal and store in the dark. (Place in a drawer at room temp.) Ensure that the solution is
covering the membrane and do not shake it. Let the color development occur in the dark
for 30-60min.

10. When the desired spots appear in sufficient intensities, stop the reaction by washing
the membrane with Buffer #4 for 5min. Let air dry and store in the dark. The spots will
fade in the light.

11. Compare the spot intensities of the control and experimental dilutions to estimate the
concentration of the experimental probe.
For example, if the spot intensity of the control spot C (10pg labeled control DNA) is
equal to the intensity of the D spot of your unknown probe which is diluted 1:1000(=103)
than calculate the amount of DIG-labeled probe DNA to be as follows:

10pg/ul X 103 = 10,000pg/ul

The total yield of the DNA labeled is the concentration of the DIG labeled probe
multiplied by the volume of the probe suspension. If labeling reaction volume was in
20ul than: 10,000 pg/ul x 20ul = 200,000pg or 200ng of labeled DNA
Use this as a guide to figure out your concentration.
Page 8

12. Proceed to the Hybridization protocol.

Solutions for Estimating Yield of Labeled Probe.

DIG Nucleic Acid Detection Kit, Boehringer Mannheim

Cat. No. 1175 041 includes:

Labeled control DNA (Cat. No.1585 738)

Anti-DIG-AP conjugate (Cat. No.1093 274)
NBT (Cat. No. 1 087 479)
X-Phosphate [BCIP] (Cat. No. 1 017 365)
Blocking Reagent (Cat. No. 1096 176). Use Nonfat Dry instead. It's much less
expensive and works the same.

These items are available separately see Cat.No. above.

Additional solutions: some are the same as the chemiluminescent detection solutions

1) Maleic Acid Buffer

Concentration Amount of Stock solution

0.1M Maleic acid 11.60g
0.15M NaCl 8.77g__________________
pH to 7.5 using NaOH pellets add slowly until pH is reached,
then add the rest of ddH20 to 1 liter.

2) WASHING BUFFER

Concentration Amount of Stock solution

Maleic Acid Buffer Make the same as above

Tween 20 0.3% 3ml for 1 Liter

3) BLOCKING STOCK SOLUTION 10X conc.

Blocking reagent from BMB, 10% (w/v) in maleic acid buffer. Dissolve blocking
reagent by constantly stirring on a heating block at (650C). Do not boil the solution. It is
difficult to get into solution and may take several hours. Be sure that all of it has
dissolved and then autoclave. Store at 40C. The solution is opaque.

A much cheaper alternative to BMB blocking reagent is non-fat dry milk. To prepare
dissolve 1g/ 100ml of maleic acid buffer. DO NOT autoclave this solution. Short term
storage at 40C ( 2-3days).
Page 9

4) BUFFER #2 (make fresh for each use)

Concentration Amount of Stock solution

1% Blocking buffer 10ml

Maleic Acid Buffer 90ml

5) DETECTION BUFFER #3 (*MgCl2 is only needed for color detection not CSPD)

Concentration Amount of Stock solution

0.1M Tris-HCl 12.1g

0.1M NaCl 5.84g
ddH2O 600ml
*50mM MgCl2 25ml of 2M stock_____
pH to 9.5
add ddH2O to 1 Liter

6) BUFFER #4

Concentration Amount of Stock solution

10mM Tris-HCl 10ml of 1M pH 8.0

1mM EDTA 2ml of 0.5M pH 8.0
ddH2O 988ml of ddH2O_____
1 Liter
Page 10

TROUBLESHOOTING

Obtain a copy of the Genius System User’s Guide for Membrane Hybridization from
Boehringer Mannheim. It contains a troubleshooting section that may help you.

The biggest problem with the Genius System is background (nonspecific binding to the
nylon membrane) which is difficult to strip off. Most often this is due to adding too
much probe to the hybridization solution.

The procedure involving quantitating the probe can be used as confirmation that your
probe has been labeled, however experience has shown that it is sometimes inaccurate for
quantitation of the probe. As a guideline the first time I use a newly labeled probe (3ug
DNA labeled O/N) I use 1ul of the labeled probe in 20ml of hybridization solution for a
15x15cm blot. This usually works well. If background is present you can:

1) Decrease amount of probe in the hybridization and test a new blot. It will be difficult
to strip the blot of the background especially if you are using a DIG labeled standard
which limits the stringency of the stripping but you can try it if you want.

2) Increase the amount of DNA in each well to 15->20ug.

3) Do both of the above.