Use the following conditions for the thermocycler:
(a) Initial denaturation step: 94
C—4 min.
(b) Denaturation: 94
C—30 s.
(c) Annealing: 62
C—30 s.
(d) Extension: 72
C—2 min (generally, 1 min for 1000 bp).
2. PCR-amplified products are visualized in an agarose gel. DNA
bands are cut out and purified. Purified fragments are cloned
into a cloning vector, according to Table 2. Incubate the reaction mixture for 1 h at room temperature.
3. After the ligation reaction, plasmids are used to transform
competent E. coli cells, which are then seeded on LB agar plates
containing 100 μg/mL ampicillin, 0.5 mM IPTG, and 80 μg/
mL X-gal for blue/white screening of recombinant bacterial
colonies with the lac
+ genotype. Plates are incubated overnight
at 37
C.
Table 1
Reagents used for PCR preparation
Recipe for PCR amplification
Reagents
Use concentration
Stock concentration
PCR buffer
1Â
10Â
MgCl 2
3 mM
50 mM
Primer
1 μM
1 0 μM
dNTPs
200 μM
1 0 m M
Taq polymerase
0.5 μL
DNA
100 ng
Pure water
Complete to a final volume 50 μL
Table 2
Recipe for preparation of a ligation reaction
Ligation reaction for insertion of a fragment into a cloning vector
Reagents
Concentration
Buffer T4 DNA ligase
1Â
T vector
50 ng
PCR product
a
T4 DNA ligase
1 μL
Pure water
Complete to a final volume 10 μL
a
Use three times more DNA insert in relation to vector
176
Aline F. Teixeira and Ana L. T. O. Nascimento