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High performance is the result of many factors:
1. 1 
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2. Very small particles of narrow distribution range makes the column densely packed.
3. Uniform pore size and distribution.
4. Accurate low volume sample injectorsr
5. Sensitive low volume detectors.

1%

1. HPLC is a fully automated process.
2. It uses high speed pump to force compounds instead of gravity. Thus it takes only a few
minutes to produce results. This is a marked difference over liquid chromatography.
3. The results produced are of a high resolution and are easy to read.
4. Tests are easily reproduced via the automated process.
5. The particles are densely packed within column.
6. Extremely sensitive but accurate procedure.

?
 ?
1. It is difficult to detect coelution (two compounds escaping from the tubing at once) with
HPLC, which may lend to inaccurate compound categorization.
2. There is a high cost for equipment needed to conduct HPLC.
3. Its operation can be complex, requiring a trained technician to operate.
4. Because of the speed of the process, the equipment has low sensitivity to some
compounds.
5. Irreversibly adsorbed compounds not detected.
Comparing HPLC and LC:
HPLC as compared with the classical LC technique is characterized by:

1. high resolution;
2. small diameter (4.6 mm), stainless steel, glass or titanium columns;
3. column packing with very small (3, 5 and 10 ȝm) particles;
4. relatively high inlet pressures and controlled flow of the mobile phase;
5. continuous flow detectors capable of handling small flow rates and detecting very small
amounts;
6. rapid analysis;
?

Types of HPLC: (LIZA sheet for simple description)

There are many ways to classify liquid column chromatography. If this classification is based on
the nature of the stationary phase and the separation process, three modes can be specified.

1. Adsorption chromatography: The stationary phase is an adsorbent (like silica gel or any
other silica based packing) and the separation is based on repeated adsorption-
desorption steps.Concerning this type, two modes are defined depending on the relative
polarity of the two phases:

a. Normal phase chromatography: The stationary bed is strongly polar in nature (e.g.
silica gel), and the mobile phase is nonpolar (such as n-hexane). Polar samples are
thus retained on the polar surface of the column packing for longer than less polar
materials.
b. Reversed-phase chromatography: The stationary bed is (nonpolar) in nature, while
the mobile phase is a polar liquid, such as mixtures of water and methanol or
acetonitrile. Here the more nonpolar the material is, the longer it will be retained.

1. Ion-exchange chromatography: The stationary bed has an ionically charged surface of

opposite charge to the sample ions. The stronger the charge on the sample, the
stronger it will be attracted to the ionic surface and thus, the longer it will take to elute.
The mobile phase is an aqueous buffer, where both pH and ionic strength are used to
control elution time.
2. Size exclusion chromatography: The column is filled with material having precisely
controlled pore sizes, and the sample is simply screened or filtered according to its
solvated molecular size. Larger molecules are rapidly washed through the column;
smaller molecules penetrate inside the porous of the packing particles and elute later.


V
V
 

 
   In plants, a breakthrough in DNA extraction came in 1980 with the
development of the CTAB protocol. CTAB is a cationic detergent that is compatible with the high
salt concentrations often used to dissociate DNA from chromosomal proteins, and can also be
used to selectively precipitate nucleic acids.

CTAB-isolated plant DNA is now routinely used for many purposes including mapping and
cloning, which have led to much of what we know about how plant genes function. It is also
widely used in the polymerase chain reaction (PCR), allowing researchers to detect small
amounts of a speci¿c sequence in complex samples. CTAB-extracted DNA is also used to
detect transgenes and to identify transgenic plants without the need for selection, to screen
plants for the presence of pathogen DNA and to screen for plants lacking certain transgenes.

The initial step in the isolation of any cellular component is breakage of the cell. Plant cell walls
present a formidable barrier to breakage, and must be enzymatically digested or mechanically
broken before extraction can occur. There are many ways to break plant cell walls. We have
had good success with methods in which frozen plant tissue is pulverized by high-speed
shaking with small metal balls in a µµball mill¶¶ or µµmixer mill.¶¶ Because breakage is very rapid,
this procedure allows the samples to remain frozen throughout the grinding step, thus
minimizing enzymatic activity. If a suitable ball mill is not available, there are alternative ways to
break the cells. One option that we describe below is to grind frozen tissue in a cold mortar and
pestle, but this option requires more time and fewer samples can be extracted simultaneously.
Regardless of the method of breakage, it is important that the frozen powder is thawed only in
the presence of heated extraction buffer to prevent possible DNA degradation.



  

1| Collect the tissue sample in 2-ml microfuge tubes and snap freeze in liquid nitrogen. Place
the samples in an ultralow freezer and store the frozen tissue at -70ËC.

2| This step can be performed by using either a Mixer Mill or a mortar and pestle. The Mixer Mill
option is preferred because less time is required, more samples can be extracted and cross-
contamination is minimized. It is important that the sample be kept frozen to reduce nuclease
activity.

  !! "   

5| Preheat extraction buffer in a 65 1C water bath during the time that the tissue in Step 2 is
ground, or before the stored ground samples are removed from the ultralow freezer.
7| Incubate at 65 1C for 30 min in an incubator or water bath and invert tubes every 5±10 min to
allow mixing.

8| Centrifuge at 13,500g for 10 min

at RT to remove nonsoluble debris.

9| Dispense 800 ml of
phenol:chloroform:isoamyl alcohol
into 2-ml microfuge tubes. Prepare
one tube per sample.

10| Transfer the supernatant from

Step 8 to the microfuge tubes with
phenol:chloroform:isoamyl alcohol
that is prepared in Step 9.

13| Carefully transfer the aqueous

(upper) layer to a new 2-ml
microfuge tube containing 800 ml
cold isopropanol (stored at 20 1C)
and mix by inverting the tube and
incubating at RT for 10 min to
precipitate the DNA.

14| Centrifuge the mixture at

13,500g for 10 min as described in
Step 8.

15| Remove the supernatant using

a micropipette and then resuspend
the pellet in 250 ml TE at RT, using
the micropipette with a fresh wide-
bore tip.

16| Pipette 2.5 ml of DNase-free

RNase into sample mixture and
incubate at 37 1C for 30 min.

17| Pipette 25 ml of 3 M NaAc and

mix. Add 600 ml of precooled
ethanol (20 1C), mix and incubate
at 20 1C for 20 min to precipitate
the DNA.

18| Centrifuge the mixture at 13,500g as described in Step 12.

19| Carefully remove the supernatant by decanting or with a micropipette and then add 500
mlofcold(20 1C) 70% (v/v) ethanol. Dislodge the pellet by quick 1±2 s vortexing, or by Àicking
with ¿nger.
20| Centrifuge at 13,500g for 10 min in a microcentrifuge at RT and remove the 70% ethanol
supernatant, taking care not to disturb the pellet.

21| Dry the DNA pellet to remove residual ethanol. There are two options for drying the DNA
pellet, which differ in the time required. Use the Speedvac option for rapidly removing the
ethanol, or use air-drying option if more time is available.

>## :

22| Resuspend the pellet either in 25 mlH2O or TE, depending on what the DNA will be used
for. If the DNA will be used directly for PCR, H2O is recommended; however, if longer-term
storage is desired, TE is recommended. Samples in TE can be stored at 4 or 20 1C. Dried
pellets of high molecular weight DNA may require an extended time to dissolve completely.

LECTURE-6

D $!  $%% & 'V



4 ( !
( ) *

  One of the major concerns of nucleic acid puri¿cation is the ubiquity of
nucleases.The minute a cell dies, the isolation of DNA turns into a race against internal
degradation. Samples must be lysed fast and completely and % +!! must inactivate
nucleases to prevent nuclease degradation. Most lysis buffers contain # ,  and
-%",(+ components. DNases are much easier to inactivate than RNases. All
materials should be  .   + !  (   /001> to .  and
s. Use only enzymes and materials guaranteed to be free of contaminating nucleases.
Where appropriate,work  (   slow down potential nuclease activity.

'( !   Large DNA molecules (genomic DNA, bacterial arti¿cial chromomoses, yeast
arti¿cial chromosomes) can be easily sheared during puri¿cation. Avoid . 2 # 
## (especially through low-volume pipette tips), and any other form of "(
 when the isolate is destined for applications that require high molecuar weight DNA.

("  ": Materials that interfere with nucleic acid isolation or down stream
applications involving the puri¿ed DNA can originate from the sample. Plants, molds, and fungi
can present a challenge because of their rigid cell wall and the presence of polyphenolic
components, which can react irreversibly with nucleic acids to create an unusable ¿nal product.
The reagents of a DNA puri¿cation method can also contribute contaminants to the isolated
DNA. Reagents that lyse and solubilize samples, such as guanidinium isothiocyanate, can
inhibit some enzymes when present in trace amounts. Ethanol precipitation of the DNA and
subsequent ethanol washes eliminate such a contaminant3 (  can also be problematic. If
you experience problems with DNA puri¿ed by a phenol-based strategy, apply (  ! "  
 
% ( #( 3 (     #  "%   "   ;
hence ,   #(    ""   !  #)  #  . A mixture of
chloroform and phenol is often employed to ""- ( %  !    ; the
chloroform reduces the amount of the DNA-containing aqueous layer at the phenol interphase.
Similar to phenol,   (  ! "  + # +"2   (   + " .  +%
(  ( %3

4 (45"-(6% !) *

The success of DNA puri¿cation is dependent on the initial quality of the sample and its
preparation.

1. Ideally start with fresh sample. Old and necrotic samples complicate puri¿cation. In the case
of plasmid preparations, cell death sets in after active growth has ceased, which can produce
an increase in unwanted by-products such as endotoxins that interfere with puri¿cation or
downstream application.

2. Process your sample as quickly as possible. When samples can¶t be immediately puri¿ed,
snap freeze the intact sample in liquid nitrogen or hexane on dry ice or store the lysed extract at
-80°C. Samples can also be freeze-dried.

3. Thorough, rapid homogenization is crucial.

4. Load the appropriate amount of sample. Too much sample can cause an increase in the
viscosity of the DNA preparation and lead to shearing of genomic DNA.

7 (%% *  5*

The integrity of your RNA is best determined by electrophoresis on a formaldehyde agarose gel
under denaturing conditions. The samples can be visualized by adding 10mg/ml of Ethidium
Bromide (EtBr) (final concentration) to the sample before loading on the gel. Compare your
prep¶s 28S rRNA band (located at approximately 5 Kb in most mammalian cells) to the 18S
rRNA band (located at approximately 2.0 Kb in most mammalian cells). In high-quality RNA the
28S band should be approximately twice the intensity of the 18S band (Figure 8.1). The most
sensitive test of RNA integrity is Northern analysis using a high molecular weight probe
expressed at low levels in the tissues being analyzed. However, this method of quality control is
very time-consuming and is not necessary in most cases. 202 Martin et al. Northern analysis is
not tolerant of partially degraded RNA. If samples are even slightly degraded, the quality of the
data is severely compromised. For example, even a single cleavage in 20% of the target
molecules will decrease the signal on a Northern blot by 20%. Nuclease protection assays and
RT-PCR analyses will tolerate partially degraded RNA without compromising the quantitative
nature of the results.
>839Assessing quality
of RNA preparation via
agarose gel electrophoresis
(() This gel shows total
RNA samples (5mg/lane)
ranging from high-quality,
intact RNA (lane 2) to almost
totally degraded RNA (lane
7). Note that as the RNA is
degraded, the 28S and 18S
ribosomal bands become less
distinct, the intensity of the
ribosomal bands relative to
the background staining in
the lane is reduced, and there
is a significant shift in their
apparent size as compared
to the size standards. ( ) This
is an autorad of the same gel
after hybridization with a
biotinylated GAPDH RNA
probe followed by nonisotopic
detection.The exposure
is 10 minutes the day after
the chemiluminescent substrate
was applied. Note that
the signal in lane 2, from
intact RNA, is well localized
with minimal smearing,
whereas the signals from
degraded RNA samples show
progressively more smearing
below the bands, or when
the RNA is extremely degraded,
no bands at all (lane
7).
 ,:


5; 5 < =V7 6 7> 
4( >,>($?*
>


RNase contamination is so prevalent, special attention must begiven to the preparation of
solutions. Solutions should be prepared in disposable, RNase-free plastic ware or in RNase-free
glassware prepared in the lab. Glassware can be made RNase-free by baking at 180°C for 8
hours to overnight, or by treating with a commercial RNase decontaminating solution.
Alternatively, RNase can be removed by filling containers with 0.1% DEPC, incubating at 37°C
for 2 hours, rinsing with sterile water and then either heating to 100°C for 15 minutes, or
autoclaving for 15minutes to eliminate RNase. Electrophoresis apparatus used for RNA analysis
can be made RNase-free by filling with a 3% hydrogen peroxide solution, incubating for 10
minutes at room temperature and rinsing with DEPC-treated water. When preparing RNase-free
solutions, wear gloves and change them often. Regardless of the method used to prepare
RNase-free solutions, keep in mind that they can easily become contaminated after preparation.
This occurs when solutions are open and used regularly, or when they are shared with others. It
is wise to prepare small volumes of solutions and aliquot larger volumes into RNase free
containers. Solutions should be clearly labeled ³RNase-free´ to avoid contamination and should
only be used with RNase free pipettes and pipette tips. Also adhere to the maxim ³when in
doubt, throw it out.´
V
  (+*
The most common method of preparing RNase-free solution is diethyl pyro carbonate (DEPC)
treatment. DEPC inactivates RNases by carboxy ethylation of specific amino acid side chains in
the protein (Brown, 1991). DEPC is a suspected carcinogen, and it should always be used with
the proper precautions.
V
, '  # *
5 
*
Most protocols specify adding DEPC to solutions at a concentration0.1%, followed by mixing
and room temperature incubation for several hours to overnight. The container lid should be
loosened for the extended incubation because a considerable amount of pressure can form
during the reaction. Finally, the solution is autoclaved; this inactivates the residual DEPC by
hydrolysis, and releases CO2 and EtOH as by-products. The EtOH by-product can combine with
trace carboxylic acid contaminates in the vessel to form volatile esters, which impart as lightly
fruity smell to the solution. This does not mean that trace DEPC remains in solution. DEPC has
a half-life of 30 minutes in water, and at a DEPC concentration of 0.1%, solutions autoclaved or
15 minutes/liter can be assumed to be DEPC-free. Be aware that increasing the concentration
of DEPC to 1% can inhibit more RNase but can also inhibit certain enzymatic reactions, so more
is usually not better.

P    !   .'(


 % != "+%' (V%+ - @' (
 A

Localization of particular sequences within genomic DNA is usually accomplished by the

transfer techniques described by Southern (1975). Genomic DNA is digested with one or more
restriction enzymes, and the resulting fragments are separated according to size by
electrophoresis through an agarose gel. The DNA is then denatured in situ and transferred from
the gel to a solid support (usually nitrocellulose filter or nylon membrane). The relative positions
of the DNA fragments are preserved during their transfer to the filter. The DNA attached to the
filter is hybridized to radiolabeled DNA or RNA, and autoradiography is used to locate the
positions of bands complementary to the probe. A sequence of 1000 bp that occurs only once in
the mammalian genome can be detected in an overnight exposure if 10 g of genomic DNA is
transferred to the filter and hybridized to a probe several hundred nucleotides in length.

93     !  "   Restriction endonucleases are used to cut high-
molecular-weight DNA strands into smaller fragments. To ensure complete digests, dilute the
digest into a larger volume (thus diluting the enzyme inhibitors) and then precipitate and
resuspend the cut DNA in the desired volume.

B3   #(  The DNA fragments are then electrophoresed on an agarose
gel to separate them by size.

/3   !  If alkaline transfer

methods are used, the DNA gel is placed into
an alkaline solution to denature the double-
stranded DNA. The denaturation in an
alkaline environment may improve binding of
the negatively charged DNA to a positively
charged membrane, separating it into single
DNA strands for later hybridization to the
probe , and destroys any residual RNA that
may still be present in the DNA.

C3' (! % ""+ Just prior to the transfer, float the Nylon membrane
on top of distilled water to
 thoroughly. A sheet of nitrocellulose (or, alternatively, nylon)
membrane is placed on the gel. Pressure or a weight is applied on top of the membrane and gel
to ensure good and even contact between gel and membrane. Buffer transfers by capillary
action from a region of high water potential to a region of low water potential (usually filter paper
and paper tissues) is then used to move the DNA from the gel on to the membrane; ion
exchange interactions bind the DNA to the membrane due to the negative charge of the DNA
and positive charge of the membrane.

D3>  !  ""+ The membrane is then baked in a vacuum or regular oven
at 80 °C for 2 hours or exposed to ultraviolet radiation which cross links (via covalent bonds)
the DNA to the membrane to permanently attach the transferred DNA to the membrane.

E3 (%+ -  The prehybridization step is required before hybridization to block non-
specific sites.For prehybridization non-specific ssDNA is added. For this purpose salmon sperm
DNA, Denhardt's solution (ficol and PVP, which are large molecules to take up space and
generate more contact; and BSA, bovine serum albumen, a non-specific protein), SDS (sodium
dodecyl sulfate), and formamide are used.

:3 V%+ -   This process relies on the ssDNA hybridizing (annealing) to the DNA on the
membrane due to the binding of complementary strands. Probing is often done with 32P labeled
ATP, biotin/streptavidin or a bioluminescent probe. The temperature at which DNA denatures is
called melting temperature. It can be calculated by: Ê
 
  .

83 4(  After hybridization, excess probe is washed from the membrane typically using
saline-sodium citrate (SSC) buffer.

F3  : After washing, blot the membrane with filter paper to remove most of the excess
moisture. The pattern of hybridization is visualized on X-ray film by autoradiography in the case
of a radioactive or fluorescent probe, or by development of color on the membrane if a
chromogenic detection method is used.

 . !  ""+

6 Capacity to bind DNA is very low

6 DNA with <400 base sufficiently binds
6 As a result of hydrophobic interaction DNA is removed from membrane easily
6 It is brittle, at 80ËC the membrane breaks
6 For high humidity it swells, due to which it needs vacuum environment

 . !% ""+ 

6 It is more durable and has higher binding capacity

6 It is efficient for transfer of DNA
6 Facile labeling of probes
6 It gives more efficient blotting

,8

 ( (+  This procedure is straightforward and provides opportunities to evaluate

progress at various points (e.g., integrity of the RNA sample and how efficiently it has
transferred to the membrane). RNA samples are first separated by size via electrophoresis
in an agarose gel under denaturing conditions. The RNA is then transferred to a membrane,
crosslinked and hybridized with a labeled probe. Northern hybridization is exceptionally versatile
in that radiolabeled or nonisotopically labeled DNA, in vitro transcribed RNA and
oligonucleotides can all be used as hybridization probes.

Despite these advantages, there are limitations associated with Northern analysis. First, if RNA
samples are even slightly degraded, the quality of the data and the ability to quantitate
expression are severely compromised. Second, a standard Northern procedure is, in general,
less sensitive than nuclease protection assays and RT-PCR. A third limitation of Northern
blotting has been the difficulty associated with multiple probe analysis. To detect more than one
message, it is usually necessary to strip the initial probe before hybridizing with a second probe.
The steps involved in

Northern analysis includes:

— RNA isolation (total or poly(A) RNA)

— Probe generation
— Denaturing agarose gel electrophoresis
— Transfer to solid support and immobilization
— Prehybridization and hybridization with probe
— Washing
— Detection
— Stripping and reprobing (optional)

     Obtaining high quality, intact RNA is a critical step in performing Northern
analysis. Although there are a great number of protocols and techniques, they all share these
common attributes:

— Cellular lysis and membrane disruption

— Inhibition of ribonuclease activity
— Deproteinization
— Recovery of intact RNA

 + =   Northern blots can be probed with radioactively or nonisotopically labeled
RNA, DNA or oligodeoxynucleotide probes. Startling differences in the signal sensitivities on
Northern blots are achieved by three methods of probe synthesis ² random-priming of DNA,
asymmetric PCR-generated DNA and in vitro transcription of RNA. While probes for Northerns
and Southerns have been historically synthesized by random-primed labeling, probes
synthesized by asymmetric PCR are 3-5 fold more sensitive than random-primed probes. RNA
probes have the added advantage that they can be hybridized and washed under more
stringent conditions, which results in lower background and fewer problems with cross-
hybridization.

   =  #(   Once RNA samples are isolated, the first step in
Northern analysis is denaturing agarose gel electrophoresis. Formaldehyde has traditionally
been used as the denaturant, although the glyoxal system has several advantages over
formaldehyde. RNA samples denatured with glyoxal may show sharper bands on Northern blots
compared to samples denatured and run in the presence of formaldehyde.

!   '   '##    "" +-   Once separated by denaturing agarose gel
electrophoresis, the RNA is transferred to a positively charged nylon membrane and then
immobilized for subsequent hybridization. The best low-tech method for agarose transfer is by a
passive, slightly alkaline, downward elution. This procedure, in comparison to upward transfer,
is much faster and therefore results in tighter bands and more signal. Alternatively,
commercially available active transfer methods (electroblotter, semidry electroblotter, vacuum
blotter, pressure blotter, etc.) can be used. Once the RNA is transferred, the membrane should
be immediately treated to crosslink the RNA. This can be done by ultraviolet light (the preferred
method) or by baking.
(%+ -    V%+ - 
(  +  Prehybridization, or blocking, is required
prior to probe hybridization to prevent the probe from coating the membrane. The ultrasensitive
Hybridization Buffer can be used for both prehybridization and hybridization. Although double-
stranded DNA probes must be denatured prior to use, RNA probes and single-stranded DNA
probes can be diluted and then added to the prehybridized blot.

4( After hybridization, unhybridized probe is removed by washing in several changes of

buffer. Low stringency washes (e.g., with 2X SSC or SSPE) remove the hybridization solution
and unhybridized probe. High stringency washes (e.g., with 0.1X SSC or SSPE) remove
partially hybridized molecules.

  If a radiolabeled probe was used, the blot can be wrapped in plastic wrap to keep it
from drying out and then immediately exposed to film for autoradiography. If a nonisotopic probe
was used, the blot must be treated with nonisotopic detection reagents prior to film exposure.

 5(  There are two common methods for immobilizing RNA on a

membrane; both work equally well. UV crosslinking is one of the most popular methods, using
either a hand-held UV lamp at short wavelength, or a commercial crosslinking device. The other
common method is baking the membrane in an oven at 80°C.

& : Shortwave UV light causes the nitrogenous bases in RNA, mostly uracil, to
become highly reactive and to form covalent linkages to amine groups on the surface of the
membrane. Care must be taken not to under or overexpose the RNA to UV light ² both of
which will decrease hybridization signals. Usually a one minute exposure with 254 nm light or
three minutes with 302 nm light is sufficient. To ensure maximum sensitivity, however, the
following experiment should be carried out.

1. Prepare a gel with five identical lanes containing 1 g of RNA each. Run the gel and
transfer it to the membrane.

2. Carefully cut the membrane into identical strips containing one lane each. Wrap these
individually in a single layer of UV-transparent plastic wrap.

3. Put the strips face down on a transilluminator (or face up if using a handheld light
source). Be sure to wear UV-opaque eyewear.

4. Expose individual strips to UV light for 30 seconds, 45 seconds, 1 minute, 2 minutes,

and 5 minutes. Be sure the strips are treated exactly as they will be during actual use,
especially the degree to which they are allowed to dry before irradiation.

5. Probe the blot for an abundantly to moderately abundantly expressed message,

according to your Northern blot protocol.

6. The strip having the highest intensity signal corresponds to the optimal exposure time for
a particular membrane with a particular UV source. This experiment should be repeated
occasionally, as the energy output of a particular device may change over time.

: Baking works by heating the membrane to drive out all water solubilizing the RNA. A
large component of RNA is its hydrophobic nucleotide bases, which make hydrophobic contacts
with aromatic groups on the membrane. This interaction is affected by heating in an oven at
80°C for 15 min. The only danger in baking is that the membrane can be damaged if the heat is
not regulated to prevent temperatures from rising much higher than 100°C.

H 4  ('.% ! (V%+ - *

Increase the amount of RNA loaded in each lane.

Up to 30 g of total RNA can be loaded in each lane.

Use poly(A) RNA instead of total RNA.

For very rare messages you may need to use poly(A) RNA instead of total RNA. 10 g
of mRNA is equivalent to 300±350 g of total RNA. (mRNA comprises only about 0.5-
3% of total RNA.)

Switch from a traditional hybridization buffer to ULTRAhyb.

ULTRAhyb Ultrasensitive Hybridization Buffer can increase the sensitivity of a blot
hybridization up to 100-fold.

If using a traditional hybridization buffer, switch from DNA to RNA probes.

RNA probes are more sensitive than DNA probes when using traditional hybridization
buffer. ULTRAhybΠUltrasensitive Hybridization Buffer, however, substantially
increases the sensitivity of DNA probes making them as sensitive as RNA probes.

Use downward alkaline capillary transfer instead of neutral capillary transfer.

Alkaline transfer nicks the RNA, increasing sensitivity by more efficiently moving RNA,
especially larger transcripts, from the gel to the membrane. Downward transfer does not
crush the gel and makes use of gravity to speed up the process.

Use an optimal hybridization temperature.

Temperatures of 42°C for DNA probes and 68°C for RNA probes usually give excellent
results in 50% formamide-based hybridization buffers. Hybridization conditions for
probes that have an unusually high GC or AT content or probes with a high degree of
mismatch with the target should be determined empirically.

Use freshly synthesized radiolabeled probes.

High specific activity probes degrade rapidly due to autoradiolysis, resulting in low
signal and/or high background.

Use high specific activity probes.

The specific activity of the probe should be at least 108 cpm/ g and preferably > 109
cpm/ g.

Increase exposure time.

Low copy number RNAs can take more than 3 days to show up using 32P-labeled
probes at -70°C with intensifying screens.