Molecular cloning of a plant gene

Ashwitha Bhat

School of Life Sciences, MAHE, Manipal 576104

The aim of the internship is to get hands-on experience while studying and understanding the science behind various methods and techniques involved in the cloning of plant genes. In the current study, ascorbate peroxidase gene is selected due to its diverse biological roles in plants. First and foremost step in the study of plant genes is to amplify the desired gene and make enough copies that can be used for further studies. The RNA isolation followed by cDNA synthesis was done prior to the polymerase chain reaction (PCR). PCR involves five core ingredients viz. template, primers (forward and reverse), dNTPs (deoxy ribo nucleotide triphosphates), DNA polymerase and buffer, and three stages viz. denaturation, annealing, and extension. The amount of the components and the conditions of each stage was optimised to get the maximum and desired amplification. Herein, initially a gradient PCR was carried out (for optimisation of annealing temperature) followed by the rePCR at specific annealing temperature. The result of the PCR was checked by running a portion of the amplified reaction in agarose gel. The desired band was purified by gel elution method. Subsequently, the ligation of eluted PCR product was carried out in a desirable cloning vector in a molar ratio of 1:3 for vector to insert. The pBluescript II SK+ vector (standard cloning vector) was selected for the study as it provides scope for both selectable (ampicillin resistance) and scorable (blue white screening) markers. Transformation was then carried out with Escherichia coli TOP 10 competent cells using heat shock method. The luria agar plates were prepared with ampicillin for selective screening and, IPTG (Isopropyl $\beta$-D-1-thiogalactopyranoside) and X-gal (also BCIG or 5-bromo-4-chloro-3-indolyl-$\beta$-D-galactopyranoside) for blue white screening. The treated cells were spread on the plates and incubated overnight at 37oC. Post incubation, the blue and white colonies were observed on the plate and white colonies were further screened for the positive clones using colony PCR. This was then followed by plasmid isolation of the cultured positive colonies and restriction digestion of the plasmid DNA by specific restriction enzyme to determine the orientation of the insert in the vector and to reconfirm the transformation with recombinant plasmid.