6.2.1 DNA Restriction Digests
Restriction digestion of plasmids and PCR products were carried out using New England Biolabs (NEB) restriction enzymes according to the manufacturers recommended conditions. Before setting up the ligations, restriction digested DNA fragments were either purified using a QIAquick PCR Purification Kit (Qiagen, 28106) or were gel purified following agarose gel electrophoresis using QIAquick Gel Extraction Kit (Qiagen, 28706).
6.2.2 Ligation
Restricted and purified vector and insert DNA fragments were incubated at a molar ratio of 1:2 or 1:3 and ligated using T4 DNA Ligase (NEB, M0202) or Quick Ligation Kit (NEB, M2200) according to manufacturer’s guidelines.
6.2.3 E. coli transformation
DH5α E. coli cells were made competent using Rubidium chloride method and stored at -80˚C. 50μl cells were thawed on ice for 5 minutes. 1-5µl (5-50ng) plasmid DNA or up to 10µl of ligation mix was added to the cells and incubated on ice for 20 minutes. The DNA-cell mixture was heat shocked at 42˚C for 90 seconds and placed back on ice for 5 minutes. 950µl of LB media was added and the cells were incubated at 37˚C with rotation for 1 hour. 10% and 90% of the cells were separately plated on LB Agar plates with appropriate antibiotic selection and incubated at 37˚C overnight.
6.2.4 dam-/dcm- Competent E. coli transformation
Commercially available methyltransferase deficient E.coli cells (NEB, C2925) were used for the growth of dam/ dcm methylation free plasmids to allow usage of methylation sensitive restriction enzymes for cleavage. 50µl cells were thawed for 5 minutes and 10-100ng plasmid DNA was added. The mixture was incubated on ice for 30 minutes followed by heat shock at 42˚C for 30 seconds according to manufacturer’s protocol. Cells were cooled down on ice for 5 min before adding 950μl of room temperature LB media. Transformations were rotated at 37°C for 1 hour. 10% and 90% of the cells were separately plated on LB Agar plates with appropriate antibiotic selection and incubated at 37˚C overnight.
6.2.5 Colony PCR (E. coli)
A single colony was carefully picked up from the transformation plate using a sterile pipette tip, patched on a new plate with the same antibiotic selection and then added to 25µl PCR reaction mix. The standard reaction set up was as follows:
Reagent 25µl Reaction Final concentration
10x NEB Taq Polymerase Buffer 2.5µl 1x NEB dNTPs (10mM each) 0.5µl 0.2mM each Forward Primer (100µM) 0.2µl 0.8µM Reverse Primer (100µM) 0.2µl 0.8µM NEB Taq Polymerase (5U/µl) 0.1µl 0.02U/µl Sterile ddH2O 21.5µl -
The standard cycling conditions for the PCR reaction were as follows:
Cycles Temperature Time
Initial Denaturation 1 95˚C 5 minutes Denaturation 30 95˚C 30 seconds Annealing 46-68˚C 30 seconds Extension 72˚C 1min/kb Final Extension 1 72˚C 7 minutes Hold 15˚C 6.2.6 Plasmid DNA preparation from E. coli
A single E. coli colony was inoculated in 5ml LB media containing appropriate antibiotic (100µg/ml Ampicillin or 50µg/ml Kanamycin) and incubated at 37˚C on a rotator overnight. Cells from 3ml of the culture were harvested by centrifugation at 13000 rpm for 1 minute in an eppendorf tube. The cell pellet was resuspended in 200µl of the resuspension buffer MP1 (100mM Tris-HCl pH7.5, 10mM EDTA, 0.2mg/ml RNAse (Sigma R6513)) followed by the addition of 200µl of the cell lysis buffer MP2 (0.2M NaOH, 1%(w/v) SDS). The tube was then mixed by inverting and incubated at room temperature for 5 minutes to allow cell lysis. 200µl of the neutralisation buffer MP3 (3M Potassium Acetate, 11.5% (v/v) Glacial Acetic Acid) was then added, mixed and incubated on ice for 5 minutes. The tube was centrifuged at 13000 rpm for 5 minutes and the supernatant was transferred to a new tube. 420µl of isopropanol was added to the supernatant, mixed and incubated at room temperature for 10 minutes. The tube was then centrifuged at 13000 rpm for 10 minutes and the supernatant was discarded. The translucent pellet was washed with 500µl of 70% ethanol and centrifuged at 13000 rpm for 5 minutes. The ethanol was completely removed from the tube and the pellet was air
dried before it was resuspended in 50µl of sterile ddH2O. 1µl of the plasmid prep
was electrophoresed on a 1% (w/v) agarose gel along with the DNA ladder (NEB N3232L) to determine the DNA concentration.
6.2.7 Site –Directed Mutagenesis
The site-directed mutagenesis was carried for in vitro substitution of a single amino acid residue in the gene of interest, using QuikChange Lightning Kit (Agilent technology). Mutagenic primers were designed specific to the desired mutation. 50-100ng of plasmid DNA with the gene of interest and 100 μM of each of the designed primers were added to the reaction mixture and a PCR reaction was set up in accordance to the manufacturer's instructions. In order to digest the parental dsDNA, the PCR product obtained was digested with Dpn I restriction enzyme at 37 °C for 5 minutes. The DpnI-treated DNA was then transform into DH5-α competent cells and was further screened for the presence of the desired mutation.
6.2.8 Error-prone PCR
Plasmid containing the gene to be mutated was purified and diluted to 100ng/μl concentration. A 10x dNTP mix was prepared that contained 2mM dATP, 2mM dGTP, 10mM dCTP and 10mM dTTP. For mutagenesis PCR, a master mix was prepared for 6x50µl reactions containing 1x NEB Taq Polymerase Buffer, 1x dNTP mix, 0.3µM each of forward and reverse primers, 5.5mM MgCl2, 0.5mM MnCl2,
10ng/µl plasmid DNA and 0.05U/µl NEB Taq Polymerase. The master mix was split into six reactions and run on a Bio-Rad C1000 thermal cycler with initial denaturation at 95˚C for 1 min, 30 cycles of denaturation (95˚C for 30 sec), annealing (59˚C for 30sec) and extension (72˚C for 1min/kb) followed by final
extension at 72˚C for 7 min. PCR products were analysed by agarose gel electrophoresis and purified using a Qiagen QIAquick PCR purification kit.