562
Mahipal Singh et al
reported in the literature to introduce plasmid DNA into the cells. These methodsinclude chemical treatment (1, 2), electroporation (3, 4), use of biolistic gun (5),polyethylene glycol (6), ultrasound (7), microwave (8) and, hydrogel (9). However,the chemical methods have attained much attention in most of the laboratories, due totheir accessibility and cost effectiveness. The physiological state of cells that enablesthem to bind and take up high molecular weight exogenous DNA is called“competence”. Uptake of free DNA by
Escherichia coli
cells which have becomecompetent by treatment of chemicals providing Ca
2+
ions followed by a heat shock pulse was first reported by Mandel and Higa (10). Subsequently, severalmodifications of this method became available for transformation of
E. coli
withplasmid DNA (1, 11-13). Among many cations tested (Ca
2+
, Mn
2+
, Sr
2+
, Ba
2+
, Mg
2+
,Na
+
and Rb
+
), Ca
2+
(100-200 mM range) provided comparatively bettertransformation of
E. coli
(14). Physiological conditions for optimum transformation,however, vary from strain to strain, their genetic background, and type of transforming DNA (14). Transformation frequencies obtained using these methodsrange approximately 10
5
-10
7
transformants/µg of DNA. The frequencies are stillabout tenfold lower when a DNA ligation reaction mixture is used as input DNA,resulting in lower recombinant clones per plate. Although for cloning and sub-cloningpurposes high efficiency of transformation is not critical, applications such asconstruction of genomic and cDNA libraries require a very high efficiency of transformation, in order to have proper representation of low copy number sequences.It is, therefore, important to optimize and improve the frequency of transformation of the desired host strain to achieve cloning of low copy number DNA molecules.
Material and Methods:
Bacterial Strain, culture media and plasmid DNA used
Escherichia coli
strain DH5
α
-T1
R
(genotype: F
-
Φ
80
lac
Z
Δ
M15
Δ
(
lac
ZYA-
arg
F)U169
rec
A1
end
A1
hsd
R17(r
k -
, m
k +
)
pho
A
sup
E44
thi
-1
gyr
A96
rel
A1
ton
A)was used in this study (Invitrogen Inc.). Luria-Bertani (LB) medium (Sigma AldrichInc) was routinely used to culture
E. coli
. For making plates medium was solidifiedwith 1.6% agar. Antibiotic plates for selection of transformants contained ampicillin(Sigma Aldrich Inc.) at a final concentration of 100 µg/ml. super coiled pUC19plasmid DNA (2686bp long) was used as a transforming DNA.
Transformation procedure
The CaCl
2
treated 50µl aliquots of competent cells stored at -80°C were thawed at icefor 30 min. Five µl of pUC19 plasmid containing a total of 50pg DNA was directlypipetted over competent cells. These cells were mixed gently by tapping 4-5 times,incubated on ice for 30 min, which was followed by a heat shock treatment. Aftertreatment the cells were routinely incubated at ice for 2 min followed by addition of 250 µl of SOC media to each vial. The vials were finally incubated at 37°C for 1 hr at225 rpm in a shaking incubator. The cultures were appropriately diluted in LBmedium and 50-100µl of each culture was plated in triplicates on ampicillin
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