Target DNA is ligated to a blunt-ended plasmid DNA, and the products of the ligation reaction
are used to transform competent E. coli. The maximum number of "correct" clones can
generally be obtained from ligation reactions containing equimolar amounts of plasmid and
target DNAs, with the total DNA concentration being <100 µg/ml. Blunt-end ligation catalyzed
by bacteriophage T4 DNA ligase is suppressed by high concentrations (5 mM) of ATP and
polyamines such as spermidine.
Protocol 20: Dephosphorylation of Plasmid DNA
During ligation in vitro, T4 DNA ligase will catalyze the formation of a phosphodiester bond
between adjacent nucleotides only if one nucleotide carries a 5´-phosphate residue and the
other carries a 3´-hydroxyl terminus. Recircularization of vector DNA can therefore be
minimized by removing the 5´-phosphate residues from both termini of the linear, double-
stranded plasmid DNA with alkaline phosphatase.
Protocol 21: Addition of Synthetic Linkers to Blunt-ended DNA
Linkers are small self-complementary pieces of synthetic DNA, usually 8-16 nucleotides in
length, that anneal to form blunt-ended, double-stranded molecules containing a restriction site.
Linkers are used to equip blunt-ended termini of DNA with restriction sites as an aid to cloning.
Protocol 22: Ligating Plasmid and Target DNAs in Low-melting-temperature Agarose
Ligation in low-melting-temperature agarose is much less efficient than ligation with purified
DNA in free solution and requires a large amount of DNA ligase. The method is used chiefly for
rapid subcloning of segments of DNA in dephosphorylated vectors and assembling
recombinant constructs.
Protocol 23: The Hanahan Method for Preparation and Transformation of Competent E.
coli: High-efficiency Transformation
This procedure generates competent cultures of E. coli that can be transformed at high
frequencies (5 x 10
8
transformed colonies/mg of superhelical plasmid DNA). IMPORTANT All
steps in this protocol should be carried out aseptically.
Protocol 24: The Inoue Method for Preparation and Transformation of Competent E.
Coli: "Ultra-Competent" Cells
This protocol reproducibly generates competent cultures of E. coli that yield 1 x 10
8
to 3 x 10
8
transformed colonies/mg of plasmid DNA. The protocol works optimally when the bacterial
culture is grown at 18°C. If a suitable incubator is not available, a standard bacterial shaker can
be set up in a 4°C cold room and regulated to 18°C.
Protocol 25: Preparation and Transformation of Competent E. coli Using Calcium
Chloride
This protocol, developed approx. 30 years ago, is used to prepare batches of competent
bacteria that yield 5 x 10
6
to 2 x 10
7
transformed colonies/µg of supercoiled plasmid DNA.
Protocol 26: Transformation of E. coli by Electroporation
Electrocompetent bacteria are prepared by growing cultures to mid-log phase, washing the
bacteria extensively at low temperature, and then resuspending them in a solution of low ionic
strength containing glycerol. DNA is introduced during exposure of the bacteria to a short high-
voltage electrical discharge.
Protocol 27: Screening Bacterial Colonies Using X-gal and IPTG: -Complementation
-complementation occurs when two inactive fragments of E. coli -galactosidase associate
to form a functional enzyme. Many plasmid vectors carry a short segment of DNA containing
the coding information for the first 146 amino acids of
-galactosidase. Vectors of this type are
used in host cells that express the carboxy-terminal portion of the enzyme. Although neither the
host nor the plasmid-encoded fragments of
-galactosidase are themselves active, they can
associate to form an enzymatically active protein. Lac
+
bacteria that result from -
complementation are easily recognized because they form blue colonies in the presence of the
chromogenic substrate X-gal. However, insertion of a fragment of foreign DNA into the
polycloning site of the plasmid almost invariably results in production of an amino-terminal
fragment that is no longer capable of -complementation. Bacteria carrying recombinant
plasmids therefore form white colonies. The development of this simple blue-white color test
has greatly simplified the identification of recombinants constructed in plasmid vectors.
Protocol 28: Screening Bacterial Colonies by Hybridization: Small Numbers
This procedure, a variant of the Grunstein and Hogness (1979) method, is used to screen a
small number of bacterial colonies (<200) that are dispersed over several agar plates and are
to be screened by hybridization to the same radiolabeled probe. The colonies are gridded onto
a master plate and onto a nitrocellulose or nylon filter laid on the surface of a second agar
plate. After a period of growth, the colonies on the filter are lysed and processed for
hybridization. The master plate is stored until the results of the screening procedure become
available.
Protocol 29: Screening Bacterial Colonies by Hybridization: Intermediate Numbers
Bacterial colonies growing on agar plates are transferred en masse to nitrocellulose filters. The
spatial arrangement of colonies on the plates is preserved on the filters. After transfer, the
filters are processed for hybridization to an appropriate radiolabeled probe while the original
(master) plate is incubated for a few hours to allow the bacterial colonies to regrow in their
original positions. This technique, a variant of the Grunstein and Hogness (1975) method, was
developed at Cold Spring Harbor Laboratory in 1975. The procedure works best with 90-mm
plates containing <2500 colonies.
Protocol 30: Screening Bacterial Colonies by Hybridization: Large Numbers
This procedure is used to plate, replicate, and subsequently screen large numbers of bacterial
colonies (up to 2 x 10
4
colonies per 150-mm plate or 10
4
colonies per 90-mm plate).
Protocol 31: Lysing Colonies and Binding of DNA to Filters
In this protocol, based on the procedure of Grunstein and Hogness (1975), alkali is used to
liberate DNA from bacterial colonies on nitrocellulose or nylon filters. The DNA is then fixed to
the filter by UV-cross-linking or baking under vacuum.
Protocol 32: Hybridization of Bacterial DNA on Filters
This protocol describes procedures to hybridize DNA from transformed colonies immobilized on
filters with radiolabeled probes and to recover from a master plate the corresponding colonies
that hybridize specifically to the probe. The method is based on the procedure published by
Grunstein and Hogness (1975).
http://www.molecularcloning.com/members/chapter.jsp?chapter=112 (2 / 3) [2002-2-18 16:10:49]
