DNA cloning

2.9.1 DNA endonuclease digestion

DNA was digested with Roche restriction endonucleases at their recommended temperatures and in their recommended buffers. The total concentration of glycerol in the reactions was kept at 5% (v/v) or below to reduce the incidence of ‘star’ activity, whereby the

endonucleases cleave additional sequences similar to their normal targets.

Typically, plasmid DNA was digested with 10 U of restriction enzyme (1 μL) in their appropriate SuRE/Cut buffer in a volume of 20 μL. Where two restriction enzymes, e.g., NotI and PstI required the same buffer, the DNA was simultaneously digested with both enzymes together. Endonuclease reactions were typically carried out at 37°C for 1-16 h depending upon whether it was for diagnostic purposes (short incubation) or for gel purification (long incubation). The DNA fragments were resolved after digestion by agarose gel electrophoresis (Section x).

2.9.2 DNA purification for cloning

PCR products and plasmids were purified for sequencing reactions and cloning either by using the High Pure PCR Product Purification Kit (www.roche-applied-

science.com) or by gel purification. Gel purification was carried out with the Zymoclean™

Gel DNA Recovery kit (Zymo Research Corporation, Irvine, CA, USA). Purification was performed to remove unused dNTPS and primers.

2.9.3 DNA quantification

DNA was quantified by gel and spectrophotometric methods. For in-gel quantification, the DNA was separated in an ethidium-bromide-containing agarose gel (section) alongside the Low DNA Mass™ Ladder or High DNA Mass™ Ladder (Life

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Technologies). The fluorescence intensity of the DNA under UV was then visually compared to fragments of known mass in the ladders. DNA was also quantified and its purity measured with a Nanodrop Spectrophotometer ND-1000 (Thermo Fisher Scientific) as described in section 2.5.2 for RNA quantitation. In this case

A260 x 501 x dilution factor = ng μL-1

1_{assumes 50 μg mL}-1 _{double stranded DNA has an A}

260 = 1.0.

2.9.4 DNA ligation

PCR products were initially TA cloned into pGEM®_{-T Easy Vector (Promega}

BioSciences, San Luis Obispo, CA, USA). Ligation reactions to generate plasmid constructs were performed by using the Rapid ligation kit (Roche) according to the manufacturers’

instructions. For directional cloning vector PgREEN0229 was digested with two different restriction endonucleases. PCR products amplified by primer induced restriction enzymatic sites were digested with the corresponding enzymes. The digested PCR products were gel purified either by using Zymoclean Gel DNA Recovery Kit (Zymo Research) or High pure PCR product purification kit (Roche). For non-directional cloning vectors were dephosphorylated with Shrimp Alkaline PHOSPHATASE (Roche), following the

manufacturer’s instructions to prevent intra-molecular ligation. For ligation the insert and vector were quantified by using spectorophotometer and the amount of insert was determined by the following formula.

ng of insert = (ng of vector × kb size of insert/ kb size of vector) × insert to vector ratio

2.9.5 Transformation of NB cells

Chemically competent NB cells were transformed with plasmids using the heat shock method. Competent cells (50 μL) were removed from the minus 80°C freezer and immediately placed on ice. When the cells thawed 10-20 μL of the ligation reaction was gently added to them and the mixture placed on ice for ~5 min. The cells were then heat- shocked at 42°C for 30 sec in a water bath and transferred back to ice for ~5 min. Depending upon the antibiotic used cells were either immediately plated (when ampicillin was used) or

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allowed to recover in LB-broth for 1 h at 37°C (when streptomycin and kanamycin were used). NB cells transformed with pGEM®_{-T Easy Vector (Promega) were selected in cultures}

or on plates containing 100 mg mL-1 _{ampicillin. NB cells transformed with pGreen0229were}

selected in media containing 50 mg mL-1 _kanamycin.

2.9.6 Transformation of GV3101::pMP90 cells

Electro-competent GV3101::pMP90 cells were transformed with approximately 0.5 μg of plasmid DNA by electroporation. Aliquots (20 μL) of freshly thawed GV3101 competent cells were gently added to 1 μL of pGreen0229 and 1μL of pSOUP (to enable efficient replication of pGreen0229in GV3101::pMP90, Hellens et al. 2000). The mixture was then carefully placed between the electrodes of a pre-chilled electroporation cuvette (BioRad Laboratories Ltd, Auckland New Zealand). Cells were electroporated at 300 V in a Cell-Porator Electroporation System (Life Technologies) following the instructions of the manufacturer and then transferred to a 14 mL bacterial culture tube containing 1 mL of LB- media for recovery at 28°C with shaking (250 rpm) for 3 h. The recovered transformed cells were then spread on LB agar plates containing 50 mg L-1 _{kanamycin (to select for}

pGreen0229), 50 mg L-1 _{rifampicin (to select for GV3101) and 20 mg L}-1 _{gentamicin (to}

select for pMP90 of GV3101).

2.9.7 Construction of plasmid constructs to complement dis58

Genomic DNA of AT4G13495 was amplified from DNA of Arabidopsis thaliana (Ler-0) using the following primers, ggcCTGCAGGTTGTGGAATAATCTCTTGTCAGG (forward primer) and cgcGCGGCCGCCCAGATAAGGCAAGTGTAGGATTC (reverse primer). To facilitate the cloning in to pGrenn 0229, a PST1 site was added to 5/ _{of forward}

primer, a NOT1 restriction site was added to the 5/ _{of reverse primer. The length of amplified}

product was 5642 bp including 1832bp DNA sequence upstream of 5/ _{region of the}

AT4G13495 gene (native promoter of the gene). The amplified gene fragment was first cloned into pGEM T Easy vector by TA cloning (58TA) (Appendix 3.5.2). The plasmid was digested with ECOR1 to identify successfully transformed plasmids. The plasmid DNA was sequenced by using M13 and M14 reverse primer from ABI Sequencing and Genotyping Services at Massey. The cloned DNA fragment was excised as NOT1-PST1 fragment and

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was cloned into corresponding site of binary vector to make 58comp construct (Hellens et al., 2000). 58comp (Appendix 3.5.3) was digested with ECOR1 restriction enzymes to confirm the presence of wild-type AT4G13495 in to the vector.