Molecular Characterization and Gene Expression Profiling ... - CUSAT

Chapter 2
10 min. for the target genes. Annealing temperature and MgCl2
concentration varied for the different genes as given in Table 2.1. The PCR
products were visualized on 1.5% agarose gel.
2.2.8. Agarose gel electrophoresis
Agarose gel (1.5 %) was prepared in 1 X TBE buffer (Tris-base -10.8 g,
0.5 M EDTA – 4 ml, Boric acid – 5.5 g, double distilled water – 100 ml, pH –
8.0). Ethidium bromide (2 µl of 1 mg / ml stock stored in dark) was added to
the melted agarose. After cooling to 45 0 C, the agarose was poured on to gel
tray and was allowed to solidify. The gel try was transferred into a buffer
tank and was submerged in 1 x TBE buffer. Ten microlitre of PCR product
was mixed with 2 µl of 6 x gel loading buffer (1 % Bromophenol blue – 250
µl, 1 % xylene cyanol–250 µl, glycerol–300 µl, double distilled water–200 µl)
and loaded onto the well. Electrophoresis was done at a voltage of 3-5
volt/cm till the bromophenol blue dye front migrate to the middle of the gel.
The gel was visualized on a UV transilluminator and documentation was
performed (BioRad).
2.2.9. Cloning of the PCR product
2.2.9.1. Ligation
PCR products were cloned onto the pGEM-T East vector (Promega,
USA). The ligation mix (10 µl) consisted of 5 µl ligation buffer (2x), 0.5 µl of
the vector (50ng/ µl), 3.5 µl PCR product (600 ng/ µl) and 1 µl of T4 DNA
ligase (3U/ µl). The ligation mix was incubated at room temperature for 1 hr.
2.2.9.2. Transformation
JM 109 high efficiency competent cells of E.coli (transformation
efficiency ≥ 1 x 10 8 cells/µg DNA), provided with the pGEM®-T Easy Vector
Systems were used for transformation. Ten microlitre of the ligated
product was mixed with 50 µl of competent cells in a polypropylene
tube and was incubated on ice for 20 min. The cells were given a heat-shock
for 45–50 seconds in a water bath at exactly 42°C without shaking, to facilitate the
Molecular Characterization and Gene Expression Profiling of Antimicrobial Peptides in Penaeid Shrimps
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