Southern blotting

Figure 2.1: Biolistic PDS-1000/He Particle Delivery system

The gun chamber was washed with 70% ethanol including macro-carrier, rupture disk, retaining cap before operation of the particle delivery system. The helium pressure was adjusted to 2000 PSI. The gold particles coated with desired plasmids were vortexed thoroughly and loaded onto the macro-carrier holder in the shooting device. The gold particles were spread over an area of about 1 cm² in the middle. A sterilized rupture disk was loaded into the sterile retaining cap and tightly secured to the end of the gas acceleration tube. The micro-carrier, macro-carrier, and stopping screening were assembled into the micro-carrier launch device. The micro-carrier launch device and targeted onion epidermal cells was inserted into the top shelf and the third shelf, respectively. The bombardment was carried out using 1100 PSI rupture disks and a vacuum of 0.1 bar. The helium bottle was closed, when all the experimental steps were finished. The helium pressure was released by applying a vacuum and “shooting” a couple of times.

2.7 Southern blotting

The principle of Southern blots (Southern, 1975) is the electrophoretical separation of DNA fragments of various sizes on an agarose gel, followed by transfer and immobilization of the DNA fragments onto a nylon membrane. The membrane-bound DNA can be then hybridized with labelled DNA probes to identify complementary sequences among the immobilized DNA.

Rupture disk

Macrocarrier

DNA coated microcarrier Stopping screen Gas Acceleration Tube

Target tissue Rupture disk

Macrocarrier

DNA coated microcarrier Stopping screen Gas Acceleration Tube

Target tissue

2.7.1 Digestion of genomic DNA with restriction enzymes

10-15 µg of genomic DNA from Arabidopsis Col-0 and T-DNA knock-out mutants were digested overnight with different restriction enzymes (150 µg): EcoRI, EcoRV, and HindIII. The sample was incubated at 37 °C overnight and additional 150 units of restriction enzyme were added and digested for another 6 to 8 hours. The completeness of the digestion was checked by electrophoresis on a 0.7 % agarose gel. The restricted DNA samples were purified by phenol: chloroform extraction (see 2.3.9), precipitated with 99.8 % ethanol at -20 °C and re-dissolved in 25 µl of dd H₂O. After supplementation with blue loading dye, the DNA samples were loaded on a 0.7 % agarose gel next to 1 µg of GeneRuler DNA ladder Mix (MBI) as size marker. The electrophoresis was run at 1 V/ cm overnight.

2.7.2 Transfer of digested genomic DNA to nylon membrane

Blotting was performed by alkaline transfer to a Hybond N⁺ nylon membrane (Amersham-Pharmacia, Freiberg, Germany). First, the gel was photographed and shortly washed in water. The upper part of the gel that contained DNA fragments > 10 kb was washed with depurination solution for 15 min. The gel was placed in denaturation (alkaline) buffer and incubated for 20 min under gentle shaking. The incubation was repeated with a fresh portion of the buffer. In the meantime, a piece of Hybond N⁺ membrane of the size of the gel was wetted with water and incubated for 20 min in denaturation buffer. Then, a capillary blot was set up as following: A plastic tray was half filled with denaturation buffer; a wick of the breadth of the gel was cut from Whatman 3 MM paper, equilibrated with denaturation buffer and placed on a platform on top of the plastic tray with its ends hanging into the buffer. The gel was placed on the wick with the DNA-side upwards, and the membrane was placed on top of the gel. The membrane was overlayed with three sheets of 3 MM paper of membrane size pre-wetted in denaturation buffer, and a stack of absorbent towels was placed on top of the 3 MM paper. The parts of the wick that were not in contact with the gel were covered with pieces of parafilm to ensure that the suction of the buffer proceeded only through the gel. Finally, a plastic plate and a weight of ca.1 kg were placed on the stack, and the transfer proceeded overnight. The membrane was washed in 2 x SSC

for 20 min for neutralization and dried at room temperature on a piece of 3 MM paper. The membrane could be stored in a plastic bag at 4 °C.

Denaturation buffer :

NaCl 1.5 M

NaOH 0.4 M

20 x SSC :

NaCl 3 M

Na3 citrate 0.3 M adjust pH7.0

2.7.3 Hybridization of DNA gel blots 2.7.3.1 Labelling of probes

For hybridization of DNA (Southern blot) immobilized on a nylon membrane, DNA probes were labelled radioactively using α-³²P-dCTP. The probes were prepared using the HexaLabel Kit from MBI Fermentas (Vilnius, Lithuania). 100 ng of DNA template (DNA fragment isolated from an agarose gel) and 10 µl of a hexanucleotide mixture provided in the kit were denatured in buffer at 100 °C for 10 min in a total volume of 40 µl. Then, 3 µl of Klenow nucleotide mixture (minus dATP), 3 µl of α-³²P-dCTP (10 µCi/ µl; Hartmann Analytik, Braunschweig, Germany) and 1 µl of Klenow exo-polymerase were added. The reaction was incubated at 37 °C for 30 min. Unincorporated α-³²P-dCTP was removed from the probes using a ProbeQuant™ G-50 Micro Columns (Amersham Pharmacia Biotech, Freiburg, Germany). The eluted probes were denatured by heating at 95 °C for 5 min and immediate chilling in ice water. The synthesized probes were ready to use.

2.7.3.2. Hybridization and washing of blots

DNA blots were placed in hybridization tubes containing ca. 15 ml of hybridization buffer and incubated at 65 °C for at least 3 hour in the hybridization oven under rotation. The labelled probes (section 2.7.3.1) were added to the hybridization tube with 15 ml fresh hybridization buffer. The hybridization proceeded at 65 °C in the hybridization oven for 16~20 hours. Afterwards, to remove non-hybridized label, the membrane was washed first twice with 200 ml of low stringent wash buffer at RT for 15 min. Then the DNA blots were washed Depurination solution :

HCl 0.125 mM

in 200 ml of high stringent buffer at 65 °C for 30 min twice. The membrane was then dried on 3MM paper, sealed into plastic bags, and the hybridization signal was detected by X-ray film at -80 °C for several days.

Low stringent buffer :

Na₂HPO₄ pH 7.2 40 mM

SDS 5 % (w/v)

EDTA 1 mM

2.8 Yeast complementation studies (in collaboration with Dr. Haslbeck,