Monarch dna gel extraction kit protocol

Thermo Scientific DreamTaq PCR Master Mix (2X) (K1071) was used for all PCR reactions. PCR products ran on a 0.7–2% agarose gel in 1X Bionic buffer (Sigma-Aldrich) to verify that the PCR reaction progressed as expected. The percentage of agarose was determined by how large a PCR product was expected. Monarch® DNA Gel Extraction Kit Protocol (NEB #T1020) was used to extract DNA from a gel slice and purify the PCR product for Sanger sequencing. Sanger sequencing was performed by Eurofins Genomics.

Purified plasmid containing cellulase gene and pET22b (+) were restricted with SacI and NdeI restriction enzymes. Restriction of the plasmid was checked with the help of 1% agarose gel electrophoresis and desired DNA fragments were purified using MONARCH DNA Gel Extraction Kit Protocol (NEB #T1020) using the manufacturer’s guide. Purified restricted cellulase was then ligated into linearized pET22b (+). The ligation mixture was incubated on ice for 1 h and then at 20 °C for overnight. Competent cells of E. coli BL21 (DE3) were then transformed with this ligation mixture containing desired heterologous plasmid by conventional heat shock method. For the screening of transformants, the transformed cells were spread over the LB-agar plates (1.5% agar containing 50 μg ml⁻¹ ampicillin) and was incubated at 37℃ overnight. A single colony was isolated and inoculated in 10 ml of LB medium and 20 ml modified M9NG media, both containing 100 µg ml⁻¹ ampicillin. Both the flasks were incubated at 37 °C, 200 rpm on shaking incubator to grow overnight.