Chapter 2: Material and Methods
2.4 Nucleic acid techniques
2.4.1 Preparation of plasmid DNA from E. coli DH5α cells
The ‘ThermoFisher GeneJET Plamid Miniprep Kit’ was used to purify plasmid DNA. A single colony of cells was used to inoculate 10 mls of LB. This was incubated at 37oC for 16 hours, shaking at 220 rpm. Cells were harvested by centrifugation at 3,000 rpm for 10 minutes (Eppendorf 5810-R centrifuge), the supernatant was discarded and plasmid DNA extracted according to the manufacturer’s instructions. DNA was eluted in 30 μl of manufacturer’s elution buffer and stored at -20oC.
2.4.2 Preparation of genomic DNA from plant leaf tissue
Approximately 1 cm2 of leaf tissue was transferred to a 1.5 ml Eppendorf tube and homogenised using a sterile pestle in 400 μl of DNA leaf extraction buffer. The mix was incubated at room temperature for 1 hour then centrifuged at 13,000 rpm for 1 minute. 300 μl of supernatant was transferred to a clean 1.5 ml Eppendorf tube then 300 μl of ice cold isopropanol was added. The mixture was vortexed and left to stand for 2 minutes to allow for DNA precipitation, before centrifugation at 13,000 rpm for 10 minutes. The supernatant was discarded and the pellet was re-suspended in 500 μl of ice cold 70% ethanol before centrifugation once more at 13,000 rpm for 5 minutes. The supernatant was discarded and the pellet allowed to completely dry. 100 μl of dH2O was used to re-suspend the pellet and typically 1-5 μl of the solution was used for PCR.
41
DNA fragments were amplified from either Hevea plant genomic/ cDNA or from a vector containing the desired sequence. Bioline’s 2x ‘ACCUZYMETM Mix’ containing ACCUZYME DNA Polymerase, MgCl2 and ultra-pure dNTPs, was generally used for PCR and reactions typically carried out in a Biometra T3000 Biocycler. A typical PCR reaction contained 1 μl (10 ng/μl) of template DNA, 1 μl each of 20 μM primer, 22 μl dH2O and 25 μl of ACCUZYMEtm Mix. A typically PCR protocol involved an initial denaturation for 3 minutes at 98oC, followed by 25 cycles of denaturation, annealing and extension at 98oC for 15 seconds, 55oC for 15 seconds and 72oC for 90 seconds/kb respectively, followed by a final extension of 2 minutes. PCR conditions varied depending on template length and complexity, and also primer melting temperature, and were optimised for each reaction. Reaction products were analysed by agarose gel electrophoresis and either excised/purified via agarose gel extraction or via PCR purification.
2.4.4 Mutation of plasmid DNA via PCR/KLD reaction
Mutations were carried out via PCR with the use of an NEB ‘Q5® Site-Directed Mutagenesis Kit.’ An initial PCR reaction was carried out using ‘Q5 Hot Start High- Fidelity 2X Master Mix’ and back-to-back mutagenic primers. A typical reaction contained 1 μl (10 ng/μl) of template plasmid DNA, 25 μl of ‘Q5 Hot Start High-Fidelity 2X Master Mix’ with 1 μl each of 10 μM primer, 9 μl of dH2O and 12.5 μl of ‘Q5 Hot Start High-Fidelity 2X Master Mix.’ A typically PCR protocol involved an initial denaturation for 20 seconds at 98oC, followed by 25 cycles of denaturation, annealing and extension at 98oC for 10 seconds, 65oC for 20 seconds and 72oC for 20 seconds/kb respectively, followed by a final extension of 2 minutes. PCR conditions varied depending on template concentration, length and complexity, and also primer melting temperature, conditions were optimised for each reaction.
Kinase, Ligase and Dpn1 (KLD) treatment, to phosphorylate and ligate the blunt ended PCR product, and also to remove template DNA, was carried out in a single reaction typically using 1 μl of PCR product, 5 μl of 2X KLD Reaction Buffer, 1 μl of 10X KLD Enzyme Mix and 3 μl of dH2O with the reaction taking place at room temperature for 5 minutes. 5 μl of KLD reaction was used to transform competent DH5α cells.
42
Initial PCR product was analysed by agarose gel electrophoresis, in the event of multiple bands the correct band was excised and purified by agarose gel purification before addition to the KLD mix.
2.4.5 Restriction endonuclease digestion of DNA
In order to prepare insert fragments and vector for ligation and to screen potential constructs for correct insertion of vector, restriction digests were performed. Using restriction endonucleases typically provided by NEB, a typical reaction contained 1 μl per restriction enzyme, 2 μl of 2X NEB CutSmart® buffer, 500 ng of DNA and up to 20 μl of dH2O. Reactions were carried out at 37oC for 1 hour followed by a 65oC heat shock for 15 minutes using a Biometra T3000 Biocycler. Reaction products were analysed using agarose gel electrophoresis and excised/purified if required via agarose gel purification.
2.4.6 Ligation of DNA fragments
DNA ligations were performed using NEB T4 ligase, following restriction digest, insert and vector were ligated at a 3:1 molar ratio for cohesive end ligations and a 5:1 molar ratio for blunt end ligations. A typical reaction consisted of 10-50 ng of vector, an appropriate amount of insert, 2 μl of 10X T4 DNA Ligase buffer, 1 μl of T4 DNA ligase and up to 20 μl volume with dH2O. Cohesive end ligations were performed at room temperature for 10 minutes and blunt end ligations were carried out at room temperature for 16 hours. Reactions were purified using a QIAGEN ‘MiniElute Reaction Cleanup Kit’ according to manufactures protocol and eluted in 10 μl of elution buffer, 2.5 μl of this was used to transform competent cells. Subsequent colonies were screen using PCR for insert and subsequent purified plasmid was screen for insert using restriction digest.
43
2.4.7 Agarose gel electrophoresis of DNA
Agarose gels were typically made with 1% (w/v) agarose dissolved in 1X TAE buffer containing GelRed Nucleic Acid Gel Stain (1 μl per 20 ml volume). DNA to load was mixed with 6X NEB Purple Loading Dye with approximately 10 μl inserted into each lane, 10 μl of Invitrogen ‘1kb’ or ‘1kb Plus’ DNA ladder diluted with dH20 and orange- G loading dye, was used as a marker. DNA was electrophoresed at 100 V until the dye reached the end of the gel and DNA was visualised using a UV trans-illuminator. 2.4.7.1 Agarose gel extraction
Visualised bands were excised from the gel using a sharp razor and transferred to 2 ml Eppendorf tubes. DNA was purified from the gel using a QIAGEN QIAquick® Gel extraction kit according to manufacturer’s instructions and eluted in 30μl of elution buffer. Purified DNA was stored at -20oC until further use.
2.4.8 Gateway cloning
Invitrogen Gateway® technology was also used to generate constructs. Primers were designed to amplify DNA fragments and attach attb1 and attb2 sites to the N’ and C’
terminals respectively. DNA fragments were purified and used directly for BP clonase reactions. DNA was cloned initially into an entry vector via a BP reaction and then subsequently transferred into the desired destination vector via an LR reaction.
2.4.8.1 BP reaction
A typical BP reaction contained approximately 150 ng of attB DNA product, 150 ng of pDonrZeo vector, 0.5 μl of BP ClonaseTM II enzyme, then made up to a total volume of 5 μl with dH20. The reaction was well mixed and then incubated either for 2 hours at 25oC or on lab bench overnight. 1 μl of Proteinase K solution was added to terminate the reaction which was then incubated at 37oC for 15 minutes. 1 μl of this reaction was transformed using DH5α cells which were plated on appropriate antibiotics. Un- recombined gateway vector was eliminated due to the presence of the lethal ccdB gene which could not grow in DH5α. Colonies were screened as described previously to check for positive entry clones.
44
A typical LR reaction contained approximately 150 ng of the desired gene in the entry vector, 150 ng of Destination vector, 0.5 μl of LR ClonaseTM II enzyme, then made up to a total volume of 5 μl with dH20. The reaction was well mixed and then incubated either for 2 hours at 25oC or on lab bench overnight. 1 μl of Proteinase K solution was added to terminate the reaction which was then incubated at 37oC for 15 min. 1 μl of this reaction was transformed using DH5α cells which were plated on appropriate antibiotics. Un-recombined destination vector was eliminated due to the presence of the lethal ccdB gene which could not grow in DH5α. Colonies were screened as described previously to check for positive clones.
2.4.9 DNA Sequencing
Sequencing of genomic, plasmid DNA or PCR products was carried out using GATC Biotech’s Sanger Sequencing service with samples prepared according to their instructions.