DNA Manipulation

2.4.1 Restriction Digestion

Plasmid DNA (250-400ng) was double digested with restriction enzymes as per NEB instructions. The digestion reaction was set up in a suitable buffer, common to both restriction endonucleases. The reaction volume was made up with water to a total of 10 μl for all digestions. The reactions were incubated at 37°C for two hours. Final concentrations of enzymes, buffers and BSA were maintained at 1/10th the total volume.

2.4.2 DNA Sequencing

DNA Sequencing was performed on a 3730xl DNA automated DNA Analyzer (Applied Biosystems) at the Biomolecular Resource Facility (BRF), JCSMR. The analyzer utilizes a high throughput capillary electrophoresis system to detect and analyze fluorescently labelled DNA sequences that were generated using the Chain Termination

principle using fluorescently labelled dideoxynucleotides (ddNTPs). A typical 20 μl reaction mix contained 2 μl of ABI PRISM Big Dye Terminator mix, 3 μl of the respective ABI Big Dye Terminator 5X sequencing buffer, 3.2pmol of primer and 300ng of plasmid DNA. The terminator mix contains fluorescently labelled ddNTPs with unlabelled ddNTPs which were incorporated into the DNA fragment during PCR amplification. A clean up with Ethanol/Sodium Acetate is required to remove residual unincorporated terminator dye which may interfere with the capillary electrophoresis procedure (Biomolecular Resource Facility DNA Sequencing Protocol). The sequencing sample is washed with 70% Ethanol twice to get rid of residual nucleotides and vacuum-dried.

Fluorescently tagged DNA fragments are run on an acrylamide gel, separating them based on size. The fluorescent tags on each of the four ddNTPs are of different emission wavelengths. A laser positioned at the bottom of the gel excites the fluorescent tag on each fragment and generates a list of nucleotides in the order of the fragment size. The nucleotides are then read off based on the wavelength of the fluorescence emitted.

2.4.3 Polymerase Chain Reaction (PCR) a. Primer Design

48 Primers were designed using NCBI Blast and Amplify 3X. Primer length was restricted to between 18-30 bps with a GC content ≤ 50% for all PCR reactions. Annealing temperatures of primer pairs were in close proximity to one another. Primers were preferably designed to end with a 3′ guanine or cytosine.

b. Cycle conditions

Cycle conditions were altered depending on the DNA template and the primer melting temperature (Tm). A single cycle denatured the DNA template at 95°C followed by

annealing of primers at the appropriate temperature which was generally the same as the lowest melting temperature of the primer pair. Extension of DNA strands by Taq Polymerase proceeded at 70°-72C°. The three steps were repeated 30-35 times and completed with a single round of extension for 5 minutes at 70°-72°C.

2.4.4 DNA Ligation

Ligation of insert with vector was carried out by T4 DNA Ligase. In the case of ligation into pGEMT, the insert was tagged with a single adenosine at both ends. An A-tailing reaction mix consisted of 0.6 μl of Taq Polymerase, 1 μl of 2 mM dATP, 0.4 μl MgCl2, 1 μl of ABI Buffer IV and 7 μl of the PCR product. The reaction was incubated at 70°C for 20 minutes. The reaction was stopped and the PCR product was purified using a PCR purification kit (Promega). 150ng of insert DNA was incubated with 50ng of vector and 1 unit of T4 DNA Ligase in a buffered reaction (total volume 10 μl) containing 1 μl of 10X T4 DNA Ligase Buffer overnight at 4°C. Insert to vector ratio was maintained at 3:1 for all ligation reactions.

2.4.5 Transformation of plasmids into bacterial cells a. Preparation of competent E.coli cells

A primary culture of E.coli in 5 ml of LB media was set up overnight at 37°C in a shaker. A secondary culture was set up by inoculating 400 ml of LB media with 2 ml of the primary culture. Cells were incubated until the absorbance at 600 nm reached 0.5- 0.9. Half of the culture was aspirated and replenished with fresh LB media. The culture was further incubated till the O.D. of 0.6 was reached. The cells were centrifuged at 4000 x g for 10 minutes at 4°C and the pellet was re-suspended in 40 ml of TfB I buffer (refer recipe in Appendix A5). Centrifugation was repeated and the cells were re- suspended in 40 ml of TfB II buffer (refer recipe in Appendix A5). Cells were then

49 aliquoted (100 μl) into sterile 1 ml microcentrifuge tubes and snap-frozen on dry ice prior to storage at -70°C.

b. Transformation of plasmids

Ligation reactions were transformed into 100 μl aliquots of competent cells and incubated on ice for 30 minutes. Cells were heat shocked at 42°C for 90 seconds and immediately allowed to recover on ice for 60 seconds. The cells were then incubated in 500 μl of LB media without antibiotics for 40 minutes. Transformed cells were then plated onto LB plates containing ampicillin and incubated overnight at 37°C.

2.4.6 Nucleic Acid Quantification

Nucleic acid concentration and purity was determined using a spectrophotometer (Nanodrop Technology, Biolab). Concentration was measured in terms of the corresponding optical density (O.D.) at a wavelength of 260 nm. Purity of DNA/RNA was estimated by calculating 260/280. A ratio of approximately 1.8 is considered pure for double stranded DNA.

2.4.7 Agarose Gel Electrophoresis

Restriction enzyme digested plasmids, PCR amplification products and colony crack PCR results were analysed by agarose gel electrophoresis. Gels varied from 0.8-1.5% in agarose concentration. Agarose was dissolved in 1X TBE Buffer (refer recipe in Appendix A5) by gradual heating. The solution was allowed to cool to 50°-60°C before adding 0.02% GelRed, a DNA binding dye that allows visualization of DNA by UV illumination. DNA samples were diluted with sucrose loading dye in a 4:1 ratio prior to loading. All gels were run in the 1X TBE buffer used to prepare the gel.