Nucleic acid techniques

2.3.1 Preparation of total RNA fromArabidopsis thalianaseeds

To isolate total RNA from Arabidopsis thaliana seeds, the protocol from Birtic & Kranner, (2006) was followed. In order to quantify the amount of RNA, samples were analysed using a Thermo Scientific NanoDrop 1000.

2.3.1.1 Reverse-transcriptase PCR

Reverse-transcriptase PCR was carried out using the Invitrogen Cloned AMV Reverse Transcriptase Kit. All quantities of RNA, reagents and PCR parameters were followed as per the manufacturer’s instructions.

2.3.2 Preparation of genomic DNA fromArabidopsis thalianaleaves

A rapid extraction protocol was utilised to isolate DNA from plant tissue which was required for PCR screening. 10-20 mg of plant leaf tissue was ground in a 1.5 ml Eppendorf tube using a 1 ml pipette tip with the end melted over as a pestle. 400 μl of extraction buffer (250 mM NaCl, 25 mM EDTA, 200 mM Tris.HCl pH 7.5, 0.5% w/v SDS) was added and the samples vortexed for 15 s and centrifuged at 20,000×g

(Eppendorf benchtop centrifuge) for 5 min. 300μl of the supernatant was transferred to a new 1.5 ml Eppendorf tube containing 300 μl of room temperature isopropanol. The samples were mixed and incubated at room temperature for 5 min. The mixture was centrifuged at 20,000×gfor 10 min and the supernatant removed by pipette. The pellet was dried on a hot block at 37°C and re-suspended in 50 μl SDW buffered to pH 8.0.

2.3.3 Amplification of DNA by conventional PCR

DNA fragments required for cloning purposes were typically amplified from plant genomic DNA or from a vector containing the desired region of DNA using conventional PCR. PCR was carried out withPwoDNA polymerase, using a Hybaid OmniGene Thermal Cycler. A typical PCR reaction mixture contained 0.5 ng template DNA, 50 pmol of each primer, 50 μM of each dNTP, 1× PCR buffer (+MgCl2) and 0.5μlPwoDNA polymerase (5 U/μl), made to a final volume of 50μl with SDW. Reactions were incubated at 94°C for 1 min and then subjected to 30 cycles of PCR (94°C (denaturing) for 15 s, 55°C (annealing) for 15 s, and 72°C (extension) for 60 s). A final extension period of 10 min at 72°C was performed and the PCR product was analysed by agarose gel electrophoresis (section 2.3.7). The times and temperatures of the PCR cycles varied depending on the size of the PCR product and the Tmof the primers.

To fuse two fragments of DNA, as opposed to inserting a restriction site between them, firstly, the two individual fragments were amplified by PCR, in the case of the first fragment, using a conventional forward primer and a reverse primer, half of which is complementary to the first fragment, and the other half complementary to the second. This technique was repeated for the second fragment, then the two

products were combined with the outer forward and reverse primers in a PCR reaction with an appropriate extension time, to yield the full-length fused construct.

2.3.4 Screening plant genomic DNA by PCR

The presence of a recombinant protein within the Arabidopsis genome of potential transgenic plants was verified using a PCR-based approach. Crude Arabidopsis

genomic DNA (section 2.3.2) was subjected to taq polymerase based PCR (30 cycles, 94°C – 15 s, 55°C – 15 s, 72 °C – 60 s, in the presence of 1.5 mM MgCl2and 0.05% W1 detergent) using gene specific primers.

2.3.5 Preparation of plasmid DNA fromEscherichia coliDH5α 2.3.5.1 Small-scale preparation of plasmid DNA

Mini-preparation of plasmid DNA was performed using the QIAprep Spin Plasmid Kit. A culture of the plasmid-containingE. coliwas prepared by inoculating 5 ml LB containing the appropriate selective antibiotic with the colony of interest and incubating at 37°C for 16 hours, shaking at 250 rpm. The following day, cells were harvested by centrifugation at 1500g (2500 rpm, Beckman GPR bench centrifuge) at 4°C for 5 minutes. The supernatant was discarded, and DNA from the resulting cell pellet was extracted according to manufacturer's protocol. The DNA was re- covered in 50 µl SDW, and either used immediately or stored at20°C.

2.3.5.2 Large-scale preparation of plasmid DNA

Large scale preparation of plasmid DNA was achieved using the QIAprep Maxi Plasmid Kit. A culture of the plasmid-containingE. coli was prepared by inoculating 2 ml LB containing the appropriate selective antibiotic with a fresh colony, and incubating at 37°C for 8 hours, shaking at 250 rpm. 1 ml of this culture was then used to inoculate 200 ml of LB containing the same concentration of the selective antibiotic, which was incubated in a sterile 1 l conical flask under the same conditions for 16 hours. Cells were harvested by centrifugation at 6000g(6000 rpm,

Beckman J2-21M/E centrifuge) at 4°C for 15 minutes. The supernatant was decanted, and the pellet was used immediately according to manufacturer's protocol. Plasmid DNA was ultimately recovered in 500 µl SDW, and stored at 20°C. DNA concentration was determined using a Thermo Scientific NanoDrop 1000.

2.3.6 Restriction endonuclease digestion of DNA

Restriction endonucleases were used to digest DNA constructs in order to confirm the insertion of DNA or prepare them for ligation (section 2.3.9). Typically, 500 ng DNA was incubated with the appropriate REact buffer to a final concentration of 1 in the presence of 1 µl enzyme (5-10 U/µl) in a total volume of 20 µl at the recommended temperature for 2 hours. Resulting DNA fragments were analysed by agarose gel electrophoresis (section 2.3.7). When more than one enzyme was used to digest DNA, if the enzymes did not have compatible REact buffers, the cut DNA was isolated and purified after agarose gel electrophoresis (section 2.3.8) between each digestion. When plasmid DNA was cut using blunt-ended restriction enzymes in preparation for the ligation of an insert, it was de-phosphorylated by incubation with 1 µl (150 U/µl) bacterial alkaline phosphatase (BAP) for 1 hour at 65°C, before being isolated and purified (section 2.3.8).

2.3.7 Agarose gel electrophoresis of DNA

Agarose gels were prepared with 0.8-2% (w/v) agarose dissolved in 1 TBE buffer (89 mM Tris, 89 mM H3BO3, 20 mM EDTA pH 8.0) containing 0.05% (w/v) ethidium bromide, to stain the DNA. DNA samples were mixed with 6 DNA sample buffer (40% (w/v) sucrose, 0.25% (w/v) bromophenol blue) to a final concentration of 1 buffer, and the mixture loaded into the wells. DNA was electrophoresed at 100 V/cm submerged in 1TBE for a suitable length of time. The DNA was visualised by placing the gels on a short wave ultra-violet trans- illuminator, and digital images recorded using a GDS8000 Documentation and Analysis System.

2.3.8 Isolation and purification of DNA from agarose gels

DNA digests and DNA amplification by PCR were carried out (sections 2.3.6 and 2.3.3 respectively) and run on agarose gels (section 2.3.7). Fragments were excised from the gel using a scalpel blade under short wave ultra-violet illumination, keeping exposure time to a minimum, and transferred to 1.5 ml Eppendorf tubes. DNA was then either stored in the gel fragment at20°C, or purified immediately, according to the manufacturer's protocol, using a QIAprep Gel Extraction Kit. DNA was typically recovered in 30 µl SDW, and either used immediately or stored at20°C.

2.3.9 Ligation of DNA fragments

DNA ligations were performed using T4 DNA ligase, either blunt-ended or following restriction endonuclease digestion and subsequent generation of sticky DNA ends (section 2.3.6). DNA fragments, typically in a 4:1 ratio of insert to vector, were mixed with 2 µl of 5T4 DNA ligase buffer (250 mM Tris.HCl pH 7.6, 50 mM MgCl2, 5 mM ATP, 5 mM DTT, 25% (w/v) polyethylene-glycol 8000) and 1 µl T4 DNA ligase (1 U/µl), and made up to a total volume of 10 µl with SDW. The mixture was normally incubated for 2 hours at room temperature or at 4°C overnight. Ligated DNA was then transformed intoE. coliDH5αcells as described in section 2.2.3.

2.3.10 Automated sequencing of plasmid DNA

Automated sequencing of constructs was performed by the Molecular Biology Service at the Department of Biological Sciences, University of Warwick, using ABI® BigDyeTM terminator chemistry and an ABI PRISM 3130xl Genetic AnalyserTM, using protocols and conditions recommended by the manufacturer. Sequencing reactions were specified to use 5.5 pmole primer and 250 ng dsDNA.