DNA Methods

Plasmid DNA isolations were purified with the JETSTAR Plasmid Purification Kit (Genomed, Bad Oeynhausen) following the instructions of the manufacturers.

PCR fragment purification was done with GFX PCR DNA and Gel Band Purification Kit (GE Biosciences) according to the manual’s instructions. DNA sequences were determined by the Sequence Core Facility at the Chair of Genome Research, Bielefeld University on the Applied Biosystems Abi Prism 3100 and 3730 sequencers using the BigDye-terminator v3.1 chemistry. Routine techniques, such as DNA agarose gel, DNA precipitation, DNA ligation, DNA cleavage with restriction endonucleases and DNA concentration measurement were done according to Sambrook and Russel, (2001).

2.4.1. Isolation of Genomic DNA

Genomic DNA was isolated according to Edwards (1991). A piece of rosette leaves, about 2 mm2, harvested from 2 to 4-weeks old Arabidopsis plants, was transferred to a 1.5ml micro centrifuge tube containing 200 μl of DNA extraction buffer. The plant sample was disrupted using a Qiagen TissueLyser. Debris was removed by centrifugation at 16000g for 8 minutes. About 150 μl of the supernatant was transferred to a new 1.5 ml micro centrifuge tube containing one volume of isopropanol. The tubes were inverted several times and incubated at room temperature for 20 minutes. The pellet was collected by centrifugation at 16000g for 5 minutes. The supernatant was discarded and the DNA was washed with 70% ethanol. The DNA pellet was air dried for 20 minutes and dissolved in 100 μl of TE buffer. This DNA was used as a template for PCR with the aim of genotyping transgenic lines.

DNA extraction buffer TE buffer

200 mM Tris/HCl pH 7,5 10 mM Tris-Cl pH 7.5 250 mM NaCl 1 mM EDTA

25 mM EDTA

0,5 % SDS

2.4.2. Polymerase Chain Reaction

Polymerase Chain Reaction (PCR) was employed to amplify DNA fragments for cloning, for genotyping of transgenic Arabidopsis plants, and for screening of transformed bacterial colonies, as well as performing site-directed mutagenic by overlap extension and 5’ rapid amplification of cDNA ends (5’RACE) used in microRNA target validation (Sambrook and Russel, 2001). General cycling

conditions were: 94o_{C to 98}o_{C for 2 minutes, 30 cycles of 94}o_{C for 30 seconds,} 55o_{C to 65}o_{C for 30 seconds and 72}o_{C (1kb/minute), and a final extension step of} 72oC for 1 minute. In this section, three protocols employed for PCR are described and another PCR-protocol variant is described in 2.5.5.2.

2.4.2.1. PCR: fragment subject to cloning

When a fragment was going to be cloned, a proof reading DNA polymerase was used. A normal reaction of 50μl contained 1X enzyme buffer, 0.2mM of each dNTP, 10 pmol of forward primer, 10 pmol of reverse primer, 1 unit of DNA polymerase, DNA template (1 μg of Matchmaker cDNA library) and water. The enzymes used were PWO DNA polymerase (Roche) and Phusion High-Fidelity DNA polymerase (Finnzymes, Finland).

2.4.2.2. Colony PCR

Colony PCR was applied whenever it was necessary to screen bacterial colonies for the presence of a desired insert. Colony PCR was done with a gene specific primer and a vector specific primer. A typical 25 μl reaction consisted of 1X Taq DNA polymerase buffer, 0.2mM of each dNTP, 10 pmol of forward primer, 10 pmol of reverse primer, 0.25 μl of Taq DNA polymerase and water. The PCR mix was distributed into reaction tubes but no template DNA was added. Instead a sterilized toothpick was used to touch a bacterial colony on a plate and then the colony was mixed with the PCR mix in each tube. Cycling conditions were almost the same the general cycling conditions, the only change was in the first step, that ws established as 95o_{C for 5 minutes.}

2.4.2.3. Overlapping PCR

To obtain miRNA binding site mutants of MYB101 and MRG1, a PCR-based site directed mutagenesis was used (Figure 7). In the first step, two separate PCRs were performed for each mutant with primers that overlap at the position (s) of the desired mutations. One pair of primers was used to amplify the DNA that contains the mutation site together with its upstream sequence. The second pair of primers was used in a separate PCR to amplify the DNA that contains the mutation site together with its downstream sequence. A third PCR was performed using the

amplicons from the previous reactions as template and a pair of primers to amplify the whole cDNA. For both genes, cDNAs previously cloned into the pMAV5-3´GFP vector were used as template.

Figure 7. Site-directed mutagenesis via overlapping PCR

Primers I to II were used to introduce point mutations at miRNA binding site region (red). Primers A and B were based on template sequence. Recovery of functional mutated amplicon was achieved by combining PCR products A-I and II-B in a single reaction with primers A and B.

2.4.2.4. Hot Stat PCR: for genotyping

In order to achieve the best results when genotyping T-DNA or transgenic lines, two parameters were changed in the basic PCR setup. ExTaq DNA polymerase (Takara, Japan) and Hot Start PCR were employed. The first reaction mix was set up in a 20 μl reaction consisting of: 1X ExTaq Buffer, 0.2mM of each dNTP, 10 pmol of forward primer, 10 pmol of reverse primer, water and 1 μl of template DNA. Tubes were placed in the thermocycler and the program was initiated. At the end of the initial denaturating step, the reaction was paused and 10 μl of the hot start mix was added (0.25 units of ExTaq DNA polymerase, 1x ExTaq buffer, water), then the cycling conditions continuined without further changes.