Standardized Southern Blot Worshop

Standardized Southern Blot Workshop Technique
A. Marcadet,' P. O'ConnelV and D. Cohen'
Establishing a Reference Protocol is sufficient for a signal) at the same time as the HLA
probe used for hybridization.
For the Southern blot section of the Tenth Histocom- As the molecular weight markers are not visible under
patibility Workshop, it was necessary to establish a UV, and because electrophoresis of DNA with identical
suitable reference protocol with reproducible, viable, conditions of agarose concentration, voltage, and run-
and simple techniques. This is the first workshop with a ning time often give different results in migration of
Southern blot section. Moreover, this protocol had to the fragments, we decided to add to the gels a marker
take account of: visible under UV, a large quantity of linearized plasmid,
1) peculiarities of the HLA system, and PAT 153.
2) organization and amount of work of each laboratory, Additional peculiarities have been introduced to this
3) necessity to compare: a) the different results (films) protocol to facilitate the work, such as special combs for
from the same laboratory, b) the results of different 30 wells. All times of experiment and quantities of
labs using the same DNAs, enzyme and probe, and c) products have been calculated and tested several times
the results from core cells and from families. with the particular conditions used for this workshop
(quantity ofloaded DNA-genomic or markers, enzymes,
These data were analyzed as follows: etc.). Twenty laboratories have tested with us different
1) The HLA system contains many genes, the products parameters such as buffers for transfer, gel loading, and
of which are highly polymorphic; the sequences of these dehybridization, binding of DNA to membranes by UV
genes are very homologous. This means that hybridiza- treatment, baking or dry air, etc.
tion with an HLA probe of restriction digests from
different genomic DNAs results, most often in many
DNA Extraction Protocol
fragments with different molecular weights, from lOObp
to 20-30Kb. To distinguish between fragments (long or Buffers and Preparation of Phenol
small) very close to each other, it was necessary to per- White cells lysis buffer: WCLB (filtered stock solution)
form for the same DNAs two electrophoresis procedures Tris pH 7.6 10 mM
(S and L) with different agarose gel concentrations, vol- EDTA pH 8 10 mM
tages, and migration times. It was also necessary to use NaCl 50 mM
many molecular weight markers from 500 bp to 30 Kb SDS 0.2%
(digests of A. phage DNA with HindIII or Kpnl or Smal Proteinase K 200 ~g/ml
or BsteII). Red cells lysis buffer: RCLB (filtered stock solution)
2) Each laboratory had to hybridize each time at least Tris pH 7.6 10 mM
six membranes and perform at least six successive MgCI, 5 mM
hybridizations. We decided to choose the labeling proce- NaCI 10 mM
dure of DNA with random primers (cheaper than "nick- Phenol: prepared on the day of use or previous day (and
translation"), and check the quality of the results obtained stored in a dark place at 4°C):
when plastic boxes (Tupperware) were used for pre- • We use phenol from Carlo Erba ref:451287 (no need
hybridization, hybridization, washing, and dehybridiza- to distill it)
tion. The efficiency for rehybridization of the chosen • Mix 3 volumes of melted phenol with 1 volume of
membrane was tested (BIOfRACE). chloroform-isoamyl alcohol (24/1), referred to here-
3) To be able to compare the films obtained with the after as PCI
same enzyme and probe, we decided to use a technique • Add 40 ml of PCI to 10 ml Tris 1M pH 8, in 50 ml
where the molecular weight markers were detected on Falcon tube mix
each membrane after each hybridization (even several • Spin 10 min at 1500 rpm/min
weeks after the first hybridization). We established and • Remove upper aqueous phase
checked a new method where a small quantity of the • Add 2ml Tris O.IM pH 8, 0.2% mercaptoethanol;
marker mix was loaded on the gels (ng amounts), trans- mix
ferred to the membrane, and then hybridized with radi- • Spin 10 min at 1500 rpm/min
oactive complementary DNA. The molecular weight • Eliminate upper aqueous phase
markers (M I and M2) were composed of a mix of digests
of A. phage DNA, and the specific probe was total A. DNA Extraction
phage DNA. This DNA was labeled (pg amounts of DNA
a) Lysis
1) From fresh peripheral blood:
Participating Laboratories: IFRACOH, 'US7LAL Collect 30 ml of blood from one individual and mix it
[;, Springer-Verlag New York 1989 553

lmmunobiology of HLA
Volume I
with 5 ml of EDTA 5% (and not with heparin, precipitation will have been achieved when the
which inhibits restriction enzymes) precipitate floats
On the day of collection, spin the mix 5 min at 1500 Put the precipitate into a 5 ml sterile Falcon tube
rpm/min Rinse three times with 4 ml 70% ethanol (do not use
Remove the upper phase of plasma without touching 100% ethanol or isopropanol)
the leukocytes Dry 5 min in a vacuum
Add red cell lysis buffer (RCLB) up to 50 ml d) Resuspension of precipitated DNA
Mix carefully 1) It is essential to wash the precipitate with 70%
Spin 10 min at 1500 rpm ethanol
Remove supernatant of lysed erythrocytes 2) Next, resuspend it in Tris 1 mM, EDTA 0.1 mM
Again add RCLB, up to 50 ml (filtered)
Spin 10 min at 1500 rpm 3) It has recently been established that resuspension is
Remove the supernatant delayed when microbubbles are present between the
Resuspend the pellet in 2 ml of RCLB precipitate and the aqueous solution.
Quickly add the white cell lysis buffer (WCLB), up to These microbubbles can be eliminated when resus-
15 ml pension is carried out in an unheated speed vacuum.
Gently agitate, horizontally, in a rotary shaker (50 Under these conditions, the DNA is resuspended in a
rpm) overnight at 42°C shorter time
Then use the protocol for the phenol-chloroform 4) If you do not have access to a speed vacuum,
extraction resuspend precipitate in a rotary shaker (slowly) for
2) From frozen peripheral blood: several hours (better at 37°C)
Sampling on EDTA (no heparin, since it inhibits res- 5) The optimal final concentration is 200 Ilglml. It is
triction enzymes) strongly recommended that you obtain equal concen-
Freeze the white blood cell pellet, obtained after two trations of all samples to assure equal loading in the
red cell lysis cycles at -80°C. Extract DNA within gel. This equal loading is essential in family studies to
1 month discriminate between homozygosity and heterozygos-
Thawing must be done quickly: place tubes in hot ity. For this purpose, it is recommended that you load
water (50°C) and leave until thawed the gel only when you are sure that homogeneity has
Quickly add WCLB, up to 15 ml, and proceed as in al) been obtained. Clearly, the initial estimate of DNA
3) From cell lines and frozen lymphocytes: concentration (before digest) is the decisive step in
Pellet 300 to 350 X 106 cells in a 50 ml Falcon tube achieving this goal. We generally check the OD of
Resuspend in 2 ml of RPMI or PBS each sample (every 24 hours) by three successive
Quickly add WCLB, up to 15 ml measurements of the sample, diluted at approximately
Gently agitate, horizontally, in a rotary shaker (50 200 Ilg/ml. This is followed by a visual check of the
rpm) overnight at 42°C DNA size by electrophoresis in 0.7% agarose gel. You
b) Phenol chloroform extraction must obtain high molecular weight DNA, greater or
Add 15 ml of PCI (1 vol) equal to A, phage, with equal amounts for each sample.
Shake 10 min in a tridimensional shaker or shake A second check is carried out after the digest during
gently by hand (a complete emulsion must be control (see below). There is a final check after migra-
obtained) tion: a photograph must be taken of each migration
Spin 3 min at 1500 rpm/min and sent to the standardization center.
Discard the lower phase of PCI with a long metal nee- e) Storage
dle (18 or 20 gauge) attached to a 50 ml glass Make aliquots of 50 Ilg
syringe; leave the interphase Store at -80°C (for several years)
Extract a second time with PCI, agitating for 5 min Keep one aliquot for use, stored for a few months at
only 4°C
Extract two times, 5 min each, with chloroform-
isoamyl alcohol 2411 (discard the lower phase of Preparation of Probes. Most of the probes used in this
chloroform after each extraction) study were cDNAs in the plasmids PBR 322 or PAT 153.
c) Isopropanol precipitation Cloning was performed by classic techniques (e. g. ,
Isopropanol allows the use of one volume of alcohol, insertion into a restriction site of the plasmid, transfor-
instead of two, with ethanol. Moreover, it does not mation of competent bacteria, selection of recombinants
precipitate RNA (precipitated RNA can lead to an on appropriate media with antibiotics).
overestimated amount of DNA when checked by Amplification of the plasmids was performed accord-
OD) ing to classical techniques, e. g. ,
Add 300 III of NaCI (3 M) to obtain a final concentra-
tion of 60 mM, followed by up to 30 ml (1 vol) of Culture of transformed bacteria
cold 100% isopropanol Addition of chloramphenicol for amplification
Agitate gently until the precipitate has formed; Lysis of bacteria (lysozyme; SDS)
554

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Standardized Southern Blot Workshop Technique

A. Marcadet,' P. O'ConnelV and D. Cohen'

[;, Springer-Verlag New York 1989 553

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