Pcr Case Study

3.2.6.2 Gel extraction of amplified PCR products

The amplified PCR product was eluted from the gel by using Quick Gel Extraction Kit (MN). The spliced gel piece was transferred to a micro centrifuge tube and added 3 volume of gel solubilization buffer to gel piece (approximately 100μl for 100mg). The sample was incubated for 10min at 50ºC water bath to dissolve the gel completely. The soluble solution was transferred to the spin column and centrifuged at 13000rpm for 2min followed by washing with 600μl washing buffer and centrifuged for 2min at 13000rpm. Then 30μl of TE buffer was added and centrifuged at 13000rpm for 2min, to get the DNA eluted from the column. Eluted DNA was checked by running on 1% agarose gel.

3.2.6.3 Ligation of PCR amplified

coli transformants was isolated by alkaline lysis method (Sambrook et. al., 1989). Clones obtained on plates were inoculated in 5ml LB broth containing appropriate antibiotic and incubated overnight at 37°C under shaking conditions. The cells were harvested by centrifugation at 7000rpm for 5min and resuspended in 200 μl of GTE (Glucose-tris-EDTA) buffer. For lysis two volumes i.e. 400 μl of SDS-NaOH (that of GTE) lysis buffer was added to the cell suspension, mixed gently and incubated at room temperature for 5min followed by adding of 200 μl pre-chilled 5M potassium acetate solution to the bacterial lysate. The lysate mixed gently by inverting the tube several times followed by incubation on ice for 10min. The mixture was centrifuged at 13,000rpm for 20min and supernatant was collected in a fresh tube. Plasmid DNA was precipitated from the supernatant by adding 0.7 volumes of isopropanol and centrifugation at 13,000rpm for 30min and washed with 70% ethanol. The pellet was air dried, resuspended in TE buffer (pH 8.0) with RNase 10μg/ml and allowed to dissolve completely. The isolated plasmid DNA was electrophoresed on 1 % agarose gel (containing Ethidium Bromide) at 75 volts and visualized under UV light in order to check its quality.

3.2.6.7 Restriction Digestion of plasmid DNA

The plasmid DNA was digested with the SalI and NotI restriction enzyme in buffer recommended by the manufacturer. Nearly 1μg of DNA was digested in a 20μl reaction volume for 3h at 37°C temperature. The reaction was terminated by heating the reaction mixture at 70°C for 10min. Restriction digestion was analyzed by agarose gel electrophoresis using DNA molecular size markers and digested product ligate into pBIND