Biology Reference
In-Depth Information
Table 5.5: Blunt-End Ligation when the Vector-to-Insert Molar Ratio
=
3.
In a siliconized Eppendorf tube in ice mix:
Dephosphorylated vector DNA (
≈
4 kb)
160 ng
DNA fragment (
≈
1 kb)
13 ng
10
×
ligase buffer I (250 mM Tris-HCl [pH 7.5], 50 mM MgCl
2
, 25% w/v polyethylene glycol
4
μ
l
8000, 5 mM DTT, 4 mM ATP)
T4 DNA ligase
1 Weiss unit
Water to a final volume of
20
μ
l
Incubate at 23°C for 4 hours and stop the reaction by adding 1
μ
l of 0.5 M EDTA.
Dilute five-fold before adding the mixture to competent
E. coli
cells for transformation.
Only T4 DNA ligase is able to join blunt-ended DNA molecules. A typical
blunt-end ligation reaction is described in
Table 5.5
.
5.8 Growth, Maintenance, and Storage of
E. coli
Escherichia coli
has been studied extensively and the genome completely
sequenced (
Snyder and Champness 1997, Neidhardt 1999
). DNA manipula-
tions require manipulating bacteria, primarily derivatives of
E. coli
K12 strains.
Different
E. coli
strains are used for different purposes (
Miller 1992
).
Standard microbial techniques are used: pure cultures of
E. coli
are obtained
by propagating cultures from single, isolated colonies on agar plates. Dilution
streaking with an inoculating loop readily produces isolated colonies and an iso-
lated colony can be restreaked to obtain a pure master plate that can be stored
at 4°C for a month. It is important that
E. coli
cultures are kept pure, the phe-
notypes are verified before use, and the cultures are stored properly. For long-
term storage, cultures can be stored in stab vials, as frozen glycerol cultures, or
as lyophilized cultures.
Overnight cultures of most strains of
E. coli
produce
≈
4
×
10
9
bacteria/ml,
depending upon the medium, degree of aeration, strain, and temperature.
To determine the cell concentration, dilutions of the culture should be plated.
Detailed methods for manipulating
E. coli
are readily available in laboratory
manuals (
Miller 1992, Sambrook and Russell 2001
).
5.9 Plasmids for Cloning in
E. coli
Plasmids are widely used as cloning vectors. Many plasmids have undergone
extensive genetic engineering to enhance their value as vectors (
Figure 5.2
).
The complete sequence of the vector usually is known, including the location of
unique restriction sites (sites where a specific endonuclease can cut the plasmid).
