Table Of ContentPreface
Recombinant DNA methods are powerful, revolutionary techniques
for at least two reasons. First, they allow the isolation of single genes in
large amounts from a pool of thousands or millions of genes. Second, the
isolated genes or their regulatory regions can be modified at will and
reintroduced into cells for expression at the RNA or protein levels. These
attributes allow us to solve complex biological problems and to produce
new and better products in the areas of health, agriculture, and industry.
Volumes 153, 154, and 551 supplement Volumes 68, 100, and 101 of
Methods ni Enzymology. During the past few years, many new or im-
proved recombinant DNA methods have appeared, and a number of them
are included in these three new volumes. Volume 351 covers methods
related to new vectors for cloning DNA and for expression of cloned
genes. Volume 154 includes methods for cloning cDNA, identification of
cloned genes and mapping of genes, chemical synthesis and analysis of
oligodeoxynucleotides, site-specific mutagenesis, and protein engineer-
ing. Volume 551 includes the description of several useful new restriction
enzymes, details of rapid methods for DNA sequence analysis, and a num-
ber of other useful methods.
YAR Wu
ECNERWAL NAMSSORG
iiix
Contributors to Volume 154
elcitrA srebmun are ni sesehtnerap gniwollof the seman of .srotubirtnoc
snoitailiffA listed era .tnerruc
TOM ALBER (27), Institute of Molecular Bi- Medicine, Stanford, California 50349
ology, University of Oregon, Eugene, Or-
D. R. DODDS (15), Sepracor, Inc., Marlbor-
egon 30479
ough, Massachusetts 25710
DANNY C. ALEXANDER (3), Calgene, Inc.,
STEPHEN ELLEDGE (7), Department of Bio-
Davis, California 95616
chemistry, Stanford University School of
K. ARAI (1), Department of Molecular Biol- Medicine, Stanford, California 50349
ogy, DNAX Research Institute of Molec-
GERALD R. FINK (10), Whitehead Institute
ular and Cellular Biology, Palo Alto, Cali-
for Biomedical Research, Cambridge,
fornia 40349
Massachusetts 02142, and Massachusetts
S. L. BEAUCAGE (15), Department of Genet- Institute for Technology, Cambridge,
ics, Stanford University, Stanford, Cali- Massachusetts 02139
fornia 50349
JOSEPH R. FIRCA (16), Pandex Laborato-
HELMUT BLOCKER (13), GBF (Gesellschaft ries, Inc., Mundelein, Illinois 60060
far Biotechnologische Forschung mbH),
D-3300 Braunschweig, Federal Republic E. F. FISHER (15), AMGen, Inc., Thousand
Oaks, California 91360
of Germany
JEF D. BOEKE (10), Department of Molecu- RONALD FRANK (13), GBF (Gesellschaftfiir
lar Biology and Genetics, The Johns Biotechnologische Forschung mbH),
D-3300 Braunschweig, Federal Republic
Hopkins University, School of Medicine,
of Germany
Baltimore, Maryland 50212
M. BROWNSTEIN (1), Laboratory of Molecu- HANS-JOACHIM FRITZ (18), Max-Planck-ln-
lar Genetics, National Institute of Child stitut fiir Biochemie, Abteilung Zellbiolo-
Health and Human Development, Be- gie, Am Klopferspitz ,81 D-8033 Mar-
thesda, Maryland 50202 tinsried bei Miinchen, Federal Republic
of Germany
PAUL CARTER (20), Department of Biomole-
cular Chemistry, Genentech, Inc., South MARK R. GRAY (8), Department of Biologi-
San Francisco, California 08049 cal Chemistry, Harvard Medical School,
Boston, Massachusetts 51120
M. H. CARUTHERS (15), Department of
Chemistry and Biochemistry, University GISELA HEIDECKER (2), Section of Genet-
of Colorado, Boulder, Colorado 80309 ics, Laboratory of Viral Carcinogenesis,
PIERRE CHAMBON (14), Laboratoire de National Cancer Institute, Frederick,
Gdndtique Moldculaire, LGME/CNRS et Maryland 10712
U.184/INSERM, Institute de Chimie LEROY HOOD (16), Division of Biology, Cal-
Biologique, Facultd de Mddecine, 67085 ifornia Institute of Technology, Pasa-
Strasbourg Cedex, France dena, California 52119
COLECLOUGH CHRISTOPHER (4), Basel Insti- SUZANNA J. HORVATH (16), Division of Bi-
tute for Immunology, CH-4005 Basel, ology, California Institute of Technology,
Switzerland Pasadena, California 52119
A. D. DARONE (15), Centocor, Inc., Mal- P. C. HUANG (22), Department of Chemis-
vern, Pennsylvania 55391 try, The Johns Hopkins University,
RONALD W. DAVIS (7), Department of Bio- School of Hygiene and Public Health,
chemistry, Stanford University School of Baltimore, Maryland 50212
ix
X CONTRIBUTORS TO VOLUME 154
MICHAEL W. (16), HUNKAPILLER Applied BRIAN W. SWEHTTAM (27), Institute of Mo-
Biosystems, Inc., Foster City, California lecular Biology, University of Oregon,
40449 Eugene, Oregon 30479
TIM (16), HUNKAPILLER Division of Biol- C. (26), MATTHEWS ROBERT Department of
ogy, California Institute of Technology, Chemistry, The Pennsylvania State Uni-
Pasadena, California 91125 versity, University Park, Pennsylvania
MITTUR N. (12), JAGADISH Division of Pro- 20861
tein Chemistry, CSIRO, Parkville 3052, GAlL P. (8), MAZZARA Applied Biotechnol-
Victoria, Australia ogy, Inc., Cambridge, Massachusetts
E. T. KAISER (25), Laboratory of 93120
Bioorganic Chemistry and Biochemistry, L. J. McBRIDE (15), Applied Biosystems,
The Rockefeller University, New York, Foster City, California 94404
New York 12001
MIHCAOJ (2), MESSING Waksman Institute
M. (1), KAWAICHI Laboratory of Molecular of Microbiology, Rutgers University, Pis-
Genetics, National Institute of Child caraway, New Jersey 08854
Health and Human Development, Be-
SAERDNA SNAHREYEM (13), GBF (Gesell-
thesda, Maryland 20205
schaft far Biotechnologische Forschung
(23), KOLLMAN PETER Department of Phar- mbH), Mascheroder Weg ,1 D-3300
maceutical Chemistry, University of Cali-
Braunschweig, Federal Republic of Ger-
fornia, San Francisco, San Francisco,
many
California 34149
C. GARRETT MIYADA (6), Department of
(18), WILFRIED KRAMER Max-Planck-Insti-
Molecular Genetics, Beckman Research
tut fiir Biochemie, Abteilung Zellbiologie,
Institute of the City of Hope, Duarte, Cal-
Am Klopferspitz ,81 D-8033 Martinsried
ifornia 91010
bei Miinchen, Federal Republic of Ger-
SEGROEG (10), NATSOULIS Department of
many
Molecular Biology and Genetics, The
SAMOHT A. KUNKEL (19), National Insti-
Johns Hopkins University, School of
tute of Environmental Health Sciences,
Medicine, Baltimore, Maryland 50212
National Institute of Health, Research
Triangle Park, North Carolina 27709 JOHN D. NOTI (12), Molecular and Cell Biol-
ogy, Triton Biosciences, Inc., Alameda,
F. LEE (1), Department of Molecular Biol-
California 10549
ogy, DNAX Research Institute of Molec-
Mar and Cellular Biology, Palo Alto, Cali- H. OKAYAMA (1),' Laboratory of Cell
fornia 94304 Biology, National Institute of Mental
Health, Bethesda, Maryland 29802
COREY LEVENSON (21), Department of
Chemistry, Cetus Corporation, Emery- DRAHCIR PINE (22), Department of Molecu-
ville, California 94608 lar and BiCoellolg y, The Rockefeller Uni-
versity, New York, New York 12001
DAVID F. (21), MARK Department of Molec-
Mar Biology, Cetus Corporation, Emery- RODNAS ROGNOP (24), Institute of Enzymol-
ville, California 94608 ogy, Hungarian Academy of Sciences, pf
7 Budapest 1502, Hungary, and Boyce
M. MATTEUCCl (15), Genentech, Inc.,
Thompson Institute, Cornell University,
South San Francisco, California 21149
Ithaca, New York 35841
HANS W. DJURHUUS MATTHES (14), La-
boratoire de Gdndtique Moldculaire, AHTIHNARP (8), REDDY Department of Biol-
ogy, Massachusetts Institute of Technol-
LGME/CNRS et U.184/INSERM, Insti-
ogy, Cambridge, Massachusetts 02139
tute de Chimie Biologique, Facult~ de
M~decine, 67085 Strasbourg Cedex, A. A. (5), REYES Department of Molecular
France Genetics, Beckman Research Institute of
CONTRIBUTORS TO VOLUME 154 Xi
the City of Hope, Duarte, California TRUEHEART JOSHUA (10), Whitehead Insti-
01019 tute for Biomedical Research, Cam-
JOHN D. STREBOR (19), National Institute of bridge, Massachusetts 02142, and Mas-
Environmental Health Sciences, National sachusetts Institute of Technology, Cam-
Institute of Health, Research Triangle bridge, Massachusetts 93120
Park, North Carolina 90772 R. BRUCE WALLACE (5, 6), Department of
MICHAEL HSABSOR (8), Department of Biol- Molecular Genetics, Beckman Research
ogy, Brandeis University, Waltham, Mas- Institute of the City of Hope, Duarte, Cal-
sachusetts 45220 ifornia 01019
KONRAD SCHWELLNUS (13), GBF (Gesell- ALICE WANG (21), Department of Molecu-
schaft fiir Biotechnologische Forschung lar Biology, Cetus Corporation, Emery-
mbH), Mascheroder Weg ,1 D-3300 ville, California 80649
Braunschweig, Federal Republic of Ger-
EGROEG M. KCOTSNIEW (9), Department of
many
Biochemistry and Molecular Biology, The
MICHAEL SMITH (17), Department of -DiB University of Texas Medical School at
chemistry, University of British Colum- Houston, Houston, Texas 52277
bia, Vancover, British Columbia
T. YOKOTA (1), Department of Molecular
MICHAEL SNYDER (7), Department of -DiB
Biology, DNAX Research Institute of Mo-
chemistry, Stanford University School of
lecular and Cellular Biology, Paid Alto,
Medicine, Stanford, California 50349
California 40349
Z. YKSNIBATS (15), Department of Chemis-
DRAHCIR A. YOUNG (7), Whitehead Insti-
try and Biochemistry, University of Colo-
tute for Biomedical Research, Cam-
rado, Boulder, Colorado 90308
bridge, Massachusetts 02142, and De-
ADRIEN STAUB (14), Laboratoire de -it~ndG partment of Biology, Massachusetts
que Mol~culaire, LGME/CNRS et U.184/
Institute of Technology, Cambridge,
INSERM, Institute de Biologique, Chimie
Massachusetts 93120
Facultd de M~decine, 67085 Strasbourg
Cedex, France DRAHCIR A. ZAKOUR (19), Molecular and
Applied Genetics Laboratory, Allied Cor-
SALGUOD SWEETSER (7), Whitehead Insti-
poration, Morristown, New Jersey 06970
tute for Biomedical Research, Cam-
bridge, Massachusetts 24120 MARK J. ZOLLER (17), Cold Spring Harbor
Laboratory, Cold Spring Harbor. New
ALADAR A. SZALAY (12), Boyce Thompson
York 42711
Institute for Plant Research, Cornell Uni-
versity, Ithaca, New York 35841 FRANS J. DE BRUIJN (ll), Max-Planck-lnsti-
J.-Y. TAN(G1 5), Shanghai Institute of -DiB tut fiir Zi~chtungsforschung, Abteilung
chemistry, Shanghai, Peoples Republic of Schell, D-5000 nlOK ,03 Federal Republic
China of Germany
JOHN W. TAYLOR (25), Laboratory of W.F. NAV GUNST (23), Department of
Bioorganic Chemistry and Biochemistry, Physical Chemistry, University of Gro-
The Rockefeller University, New York, ningen, 9747 AG Groningen, ehT Nether-
New York 12001 lands
[1] cDNA 3
CONSTRUCTION AND SCREENING OF LIBRARIES
[1] High-Efficiency Cloning of Full-Length cDNA;
Construction and Screening of cDNA Expression Libraries
for Mammalian Cells
By H. ,AMAYAKO M. ,IHCIAWAK M. ,NIETSNWORB F. ,EEL
T. YOKOTA, and K. ARAI
cDNA cloning constitutes one of the essential steps to isolate and
characterize complex eukaryotic genes, and to express them in a wide
variety of host cells. Without cloned cDNA, it is extremely difficult to
define the introns and exons, the coding and noncoding sequences, and
the transcriptional promoter and terminator of genes. Cloning of cDNA,
however, is generally far more difficult than any other recombinant DNA
work, requiring multiple sequential enzymatic reactions. It involves in
vitro synthesis of a DNA copy of mRNA, its subsequent conversion to a
duplex cDNA, and insertion into an appropriate prokaryotic vector. Due
to the intrinsic difficulty of these reactions as well as the inefficiency of
the cloning protocols devised, the yield of clones is low and many of
clones are truncated.1
The cloning method developed by Okayama and Berg 2 circumvents
many of these problems, and permits a high yield of full-length cDNA
clones regardless of their size. 6-3 The method utilizes two specially engi-
neered plasmid DNA fragments, "vector primer" and "linker DNA." In
addition, several specific enzymes are used for efficient synthesis of a
duplex DNA copy of mRNA and for efficient insertion of this DNA into a
plasmid. Excellent yields of full-length clones and the unidirectional in-
sertion of cDNA into the vector are the result. These features not only
facilitate cloning and analysis but are also ideally suited for the expression
of functional cDNA.
To take full advantage of the features of this method, Okayama and
A. Efstratiadis and L. Villa-Komaroff, in "Genetic Engineering" (J. K. Setlow and A.
HoUaender, eds.), Vol. ,1 p. .1 Plenum, New York, 1979.
2 H. Okayama and P. Berg, Mol. Cell. Biol. 1, 161 (1982).
3 D. H. Maclennan, C. J. Brandl, B. Korczak, and N. M. Green, Nature (London) 316, 696
(1985).
4 L. C. Kun, A. McClelland, and F. H. Ruddle, Cell 37, 59 (1984).
5 K. Shigesada, G. R. Stark, J. A. Maley, L. A. Niswander, and J. N. Davidson, Mol. Cell.
Biol. 5, 1735 (1985).
6 S. M. Hollenberg, C. Weinberger, E. S. Ong, G. Cerelli, A. Oro, R. Lebo, E. B. Thomp-
son, M. G. Rosenfeld, and R. M. Evans, Nature (London) 318, 635 (1985).
Copyright © 1987 by Academic Press, Inc.
METHODS IN ENZYMOLOGY, VOL. 451 All rights of reproduction in any form reserved.
4 SDOHTEM ROF GNINOLC eDNA ]1[
Berg 7 have modified the original vector. The modified vector, pcD, has
had SV40 transcriptional signals introduced into the vector primer and
linker DNAs to promote efficient expression of inserted cDNAs in mam-
malian cells. Construction of eDNA libraries in thep cD expression vector
thus permits screening or selection of particular clones on the basis of
their expressed function in mammalian cells, in addition to regular screen-
ing with hybridization probes.
Expression cloning has proven extremely powerful if appropriate
functional assays or genetic complementation selection systems are avail-
able. 41-8 In fact, Yokota et al. 21,11 and Lee et al. 41,31 have recently isolated
full-length eDNA clones encoding mouse and human lymphokines with-
out any prior knowledge of their chemical properties, relying entirely on
transient expression assays using cultured mammalian cells. Similar mod-
ifications have been made to promote the expression of cDNA in yeast,
thereby permitting yeast mutant cells to be used as possible complementa-
tion hosts.tS,16
In this chapter, we describe detailed procedures for the construction
of full-length cDNA expression libraries and the screening of the libraries
for particular clones based on their transient expression in mammalian
cells. Methods for library transduction and screening based on stable
expression are described in Vol. 151 of Methods in Enzymology. If ex-
pression cloning is not envisioned, the original vector 2 or one described
by others 71 can be used with slight modifications of the procedure de-
scribed below.
7 H. Okayama and P. Berg, Mol. Cell. Biol. 2, 280 (1983).
s D. H. Joly, H. Okayama, P. Berg, A. C. Esty, D. Filpula, P. Bohlen, G. G. Johnson, J. E.
Shivery, T. Hunkapiller, and T. Friedmann, Proc. Natl. Sci. Acad. U.S.A. 80, 477 (1983).
9 D. Ayusawa, K. Takeishi, S. Kaneda, K. Shimizu, H. Koyama, and T. Seno, J. Biol.
Chem. 259, 1436 (1984).
01 H. Okayama and P. Berg, Mol. Cell. Biol. 5, 1136 (1985).
11 T. Yokota, F. Lee, D. Rennick, C. Hall, N. Arai, T. Mosmann, G. Nabel, H. Cantor, and
K. Aral, Proc. Natl. Acad. Sci. U.S.A. 81, 1070 (1985).
21 T. Yokota, N. Arai, F. Lee, D. Rennick, T. Mosmann, and K. Arai, Proc. Natl. Acad.
Sci. U.S.A. 82, 68 (1985).
31 F. Lee, T. Yokota, T. Otsuka, L. Gemmell, N. Larson, L. Luh, K. Arai, and D. Rennick,
Proc. Natl. Acad. Sci. U.S.A. 82, 4360 (1985).
41 F. Lee, T. Yokota, T. Otsuka, P. Meyerson, D. Villaret, R. Coffman, T. Mosmann, D.
Rennick, N. Roehm, C. Smith, C. Zlotnick, and K. Arai, Proc. Natl. Acad. Sci. U.S.A.
83, 2061 (1986).
51 G. L. McKnight and B. C. McConaughy, Proc. Natl. Acad. Sci. U.S.A. 80, 4412 (1983).
61 A. Miyajima, N. Nakayama, I. Miyajima, N. Arai, H. Okayama, and K. Arai, Nucleic
Acids Res. 12, 6639 (1984).
71 D. C. Alexander, T. D. McKnight, and B. G. Williams, Gene 31, 79 (1984).
[1] CONSTRUCTION AND SCREENING OF cDNA LIBRARIES 5
Methods
Clean, intact mRNA is prepared from cultured cells or tissue by the
guanidine thiocyanate method 81 followed by two cycles of oligo(dT)-
cellulose column chromatography. The purified mRNA is then reverse
transcribed by the avian myeloblastosis enzyme in a reaction primed with
the pcD-based vector primer, a plasmid DNA fragment that contains a
poly(dT) tail at one end and a HindlII restriction site near the other end
(Figs. 1 and 2). 7 The vector also contains the SV40 poly(A) addition signal
downstream of the tail site as well as the pBR322 replication origin and
the/3-1actamase gene. Reverse transcription results in the synthesis of a
cDNA: mRNA hybrid covalently linked to the vector molecule (Fig. 3).
This product is tailed with oligo(dC) at its 3' ends and digested with
HindlII to release an oligo(dC) tail from the vector end and to create a
HindlII cohesive end. The C-tailed cDNA : mRNA hybrid linked to the
vector is cyclized by addition of DNA ligase and a pcD-based linker
DNA--an oligo(dG)-tailed DNA fragment with a HindlII cohesive end
(this linker contains the SV40 early promoter and the late splice junctions)
(Figs. 1 and 2). Finally, the RNA strand is converted to DNA by nick-
translation repair catalyzed by Escherichia coli DNA polymerase I,
RNase H, and DNA ligase. The end product, a closed circular cDNA
recombinant, is transfected into a highly competent E. coli host to estab-
lish a cDNA clone library.
In the steps that have just been enumerated, double-stranded, full-
length DNA copies of the original mRNAs are efficiently synthesized and
inserted into the vector to form a functional composite gene with the
protein coding sequence derived from the cDNA and the transcriptional
and RNA processing signals from the SV40 genome. To screen for or
select a particular cloneo n the basis of the function it encodes, the library
is acutelyt ransfectedo r stably transduced into cultured cells. Procedures
for stable transduction are described in Chap. [32] of Vol. 151 of Methods
in Enzymology.
Preparation of mRNA
Successful construction of full-length cDNA libraries depends heavily
on the quality of the mRNA preparation. The use of intact, uncontami-
nated mRNA is essential for generating full-length clones. Messenger
RNA prepared by the guanidine thiocyanate method 81 satisfies the above
81 j. M. Chiigwin, A. E. Przybyla, R. J. MacDonald, and W. J. Rutter, yrtsimehcoiB 18, 5294
(1978).
6 METHODS FOR CLONING cDNA [1]
IN.O( 1519,01 im junctioe
•ell Hiadlll
-7"/ ~ C/at ~ Xhol ,. §o7.0,1
.v, "'°2'2('
Hindlll
(0.71)
FBR322 ori
pBR~2
orl
- /f
o
I ~.~
llldniH
laS I i noO4VS / "~ ,,~'~
x~l
a#l
Awll
ltsP
pcO-X
X(©DNA)
8P ~ ,~ 4 ~ t"
~1 & IHmm8
p~A
FIG. I. Structure and component parts of the pcD vector and its precursor plasmids,
pcDV1 and pLl. The principal elements of the pcD vector are a segment containing the
SV40 replication origin and the early promoter joined to a segment containing the 19 S and
61 S SV40 late splice junctions (hatched area); the various cDNA inserts flanked by dG/dC
and dA/dT stretches that connect them to the vector (solid black area); a segment containing
the SV40 late polyadenylation signal [poly(A)] (stippled area); and the segment containing
the pBR322/3-1actamase gene and the origin of replication (thin and open area), pcDVl and
pL 1 provide the pcD-based vector primer and linker DNA, respectively. For the preparation
of the vector primer and linker DNA, see Methods sections and Fig. 2.
[1] CONSTRUCTION DNA GNINEERCS FO cDNA SEIRARBIL 7
] VECTOR PRIMER I I OLIGO (dO) TAILED LINKER DNA ]
)59.0(
1J]dniH )68.0(
ItsP S61 ecilps
~ noitcnuj
. Eco IR )857.0(
pmA R // (0.93)~ X'h^' ~ )57.0{
AmpR(/// IVDcp "~ )457.0, f/
PstI-~ ]Lp ~ 04VS iro
~ InpK--~.)17.0(
)517.0(
\\ IHmaB
(0.14~!~ XhoI 8
~ ~ "polylA) ~~~./ dniH" ]]l )17.0(
l91.0(" ~
PstI NOITSEGID
KpnI NOITSEGID
OGILO )Gd( SLIAT
YLOP )Td( SLIAT
/...-~ G G G
Igd• niH ocE
IR
~ ~ npK I
~ ~ /\ dniH IH
Eco IR NOITSEGID I]ldniH NOITSEGID
NOITACIFIRUP NOITACIFIRUP
dniH ]]]
~ T OCE ohX I
IR
TTTT dniH nI
Pstl
G
Xho I ~ Bam HI ~a
.GIF .2 Preparation of linker and vector primer .sAND
criteria and is reverse transcribed efficiently. It has successfully been
used for cloning a number of cDNAs? 41- The method described below is a
slight modification of the original method that ensures complete inactiva-
tion of RNases through all the steps of RNA isolation.
8 METHODS FOR CLONING cDNA [1]
ANRm
. . . . ~ :GNILAENNA ~. Cc c .~
ANDc ~ - OGILO
,- % ~ SYNT.ES,S 'T~ SL,AT A
,,c _
[ f T ~ ~/ A.TT - ~ffll k T
Hindlll
Hindlll~Xh°l ~ lHmaB ~
REMIRP AC C C
,sP I f2 t "~ -- "~-AA /H~m
H/ndlll ~GG G ¢/~ ,...~- -T T A J NOITSEGID
mdniH lHmaB8 [ohX
GNILAENNA OGIL OFO Gd '
DELIAT ;REKNIL NOITAZILCYC
HTIW AND//oc.E ESAGIL /
~.G~..~ __~- ~ TNEMECALPER AN RFO STRAND ~ GGc~'~--
AND ESAREMYLOP AND 1 "~" A tl~
"~~: ~ ~ AND ESAGU ]Hd~'kH --'P~ AAT
H/ndln
.GIF .3 citamyznE steps ni eht noitcurtsnoc of pcD-cDNA .stnanibmocer -angised ehT
snoit of eht AND stnemges era sa described ni Fig. .1 For latnemirepxe sliated dna -moc
,stnem see .sdohteM
stnegaeR
All solutions are prepared using autoclaved glassware or sterile dis-
posable plasticware, autoclaved double-distilled water and chemicals of
the finest grade. Solutions are sterilized by filtration through Nalgen 0.45
/zm Millipore filters and subsequently by autoclaving (except as noted). In
general, treatment of solutions with diethyl pyrocarbonate is not recom-
mended since residual diethyl pyrocarbonate may modify the RNA, re-
sulting in a marked reduction in its template activity.
5.5 M GTC solution:
5.5 M guanidine thiocyanate (Fluka or Eastman-Kodak), 25 mM
sodium citrate, 0.5% sodium lauryl sarcosine. After the pH is ad-
justed to 7.0 with NaOH, the solution is filter-sterilized and stored
at 4 .° Prior to use, 2-mercaptoethanol is added to a final concentra-
tion of 0.2 M.
4 M GTC solution: 5.5 M solution diluted to 4 M with sterile distilled
water.
CsTFA solution:
cesium trifluoroacetate (density 1.51 - 0.01 g/ml), 0.1 M ethylene-
diaminetetraacetic acid (EDTA) (pH 7.0). Prepared with cesium
trifluoroacetate (2 g/ml) (CsTFA, Pharmacia) and 0.25 M EDTA
(pH 7.0).
