Molecular Cloning

The following plasmid vectors were used for molecular cloning in this work:

pTM1, pCR3.1, pBluescript-BRSV (pBRSV) and pCR 2.1.

4.7.3.1 Cleavage of DNA with restriction enzymes

Restriction endonucleases are bacterial enzymes that recognize specific 4- to 8-bp sequences (restriction sites), and cleave both DNA strands at this site. In order to insert a foreign sequence in a plasmid vector both DNA molecules were treated with appropriate restriction enzymes. Different temperatures and incubation times were used in order to meet the particular reaction conditions. For digestion of plasmid DNA, 2-10 µg were used and the reaction was performed for 1-3 hours with 1-2 units of the respective enzyme. PCR fragments were treated with 2 units of enzyme for 16-18 hours.

4.7.3.2 Recovery of DNA from agarose gels

DNA molecules cleaved with endonucleases were separated by agarose electrophoresis. A gel containing 0.8% agarose in TAE buffer was prepared. The DNA samples were mixed with gel-loading buffer and loaded into the slots of the gel as follows: in the first slot- a molecular DNA marker was added. The second received an aliquot of the sample, the third slot was left free, and into the next couple of slots

the remaining sample was applied. The electrophoresis was carried out at 80V for 1 hour. The first part of the gel containing the molecular marker and one slot with sample was carefully cut with a scalpel and stained with a solution of ethidium bromide for 5 min. The stained gel was illuminated with ultraviolet light and the regions of the gel containing the DNA fragments were marked. The corresponding region was cut from the unstained part of the gel and collected in tubes. The extraction of the DNA fragments was performed with the QIAquick gel extraction kit or the QIAex II gel extraction kit (Qiagen). DNA concentration was photometrically determined at 260 nm.

4.7.3.3 Ligation

The purified PCR fragment was ligated with the treated purified plasmid DNA under the following condition:

1. PCR DNA fragment 7-15 ng

2. vector DNA 1-5 ng

2. 10x ligase buffer 1-4 µl 3. Bacteriophage T4 DNA ligase 1-4 µl

4. H2O to 10-40 µl

A molar ratio of plasmid vector to PCR DNA fragment of 1:1 to 1:6 was used.

The ligation reaction was performed for 1 hour at room temperature or for 16-18 hours at 14°C.

4.7.3.4 Preparation of competent E. coli

Chemicompetent E. coli

E.coli XL-1Blue were used for preparation of chemically competent bacteria.

A single bacterial colony was picked from an agar plate and propagated in 50 ml of LB medium overnight at 37°C with vigorous agitation. The overnight culture was used to inoculate 1l of LB medium which was incubated for 3 hours at 37°C with agitation.

When the optical density at 600 nm reached 0.5 the bacterial cells were transferred to 50 ml centrifugation tubes and cooled on ice for 10 minutes. The cells were pelleted by centrifugation at 2800 g for 10 minutes at 4°C and resuspended in 12.5 ml ice-cold CaCl2 buffer. After centrifugation as above the cell pellet was resuspended in 2.5 ml of ice-cold CaCl2 buffer. The cell sediment was aliquoted and frozen in liquid nitrogen and stored at –80°C.

Electrocompetent bacteria

E.coli XL-1Blue were used for preparation of electrocompetent bacteria. A single colony of E. coli from a was picked from an agar plate and propagated in 50 ml of LB medium overnight at 37°C with vigorous agitation. The overnight culture was used to inoculate 1l of LB medium which was incubated at 37°C with agitation. When the OD600 of the culture reached 0.4, the flasks was transferred to an ice-water bath for 15 minutes. Bacteria were harvested by centrifugation for 15 minutes at 4°C. The cell pellet was resuspended in 1 l of ice-cold H2O and again subjected to centrifugation. The supernatant was decanted and the cells were resuspended in 500 ml of ice-cold 10% glycerol. The cells were sedimented by centrifugation and resuspended in 20 ml of ice-cold 10% glycerol, and again pelleted by centrifugation.

The pellet was resuspended in 2 ml of ice-cold 10 % glycerol. The cell sediment was aliquoted and frozen in liquid nitrogen and stored at –80°C.

4.7.3.5 Transformation of E. coli

Transformation of E. coli by electroporation

Electrocompetent cells were thawed on ice. Plasmid DNA in a volume of 1-2 µl was added (10 pg to 25 ng) to the bacteria. The electroporation apparatus was set to 25 µF capacitance, 2.5 kV voltage, and 200 Ohm resistance. The DNA/cell mixture was transferred into a pre-cooled electroporation cuvette. The dry cuvette was placed in the electroporation device and a pulse of electricity was delivered to the cells for 4-5 milliseconds with a field strength of 12.4-5 kV/cm. After the pulse, the electroporation cuvette was removed and 1 ml of LB medium was added at room temperature. The cells were transferred to a polypropylene tube and the cultures were incubated for 1 hour at 37°C with rotation. A volume of 100 µl from the transfected bacteria were plated onto LB agar containing ampicillin (50µg/ml) and incubated for 16-18 hours at 37°C.

Heat-shock transformation of E. coli

Plasmid DNA (1-50ng) was added to 100 µl of chemicompetent E. coli and incubated on ice for 30 min. The bacteria were heated at 42 °C for 30 sec in a water bath. The tubes were rapidly transferred to an ice bath and cooled for 5 min. 250 µl SOC or LB medium was added and the cultures were incubated for 30-60 min at 37°C to allow the bacteria to recover and to express the antibiotic resistance marker encoded by the plasmid. 100 µl of the culture were plated onto LB agar medium containing ampicillin and incubated for 16-18 hours at 37°C.

4.7.3.6 Colony PCR

To test for the presence of recombinant plasmid, colonies of transfected E. coli were analyzed by PCR using appropriate primers. A PCR master mix was prepared of which each PCR tube received 15µl.

For each reaction the following reagents were used:

1. H2O 12.2 µl

3. dNTPs (10mM) 0.3 µl 4. forward primer (10 µM) 0.45 µl 5. reverse primer (10 µM) 0.45 µl 6. Taq DNA polymerase 5 units/µl 0.1 µl

A single E. coli was picked with a sterile pipette tip and dispersed first into the reaction mix and then into a 1.5 ml tube containing 250 Ml of LB-medium. The PCR reaction was performed under the following conditions:

1 cycle 95°C 2 min denaturation

95°C 15 sec denaturation 20 cycles 56°C (-0.2) 30 sec annealing

72°C 60 sec polymerization

1 cycle 72°C 5 min polymerization

The PCR products were run on an agarose gel. The cultures of two positive clones were used to inoculate 50 ml of LB medium.

4.7.3.7 Plasmid DNA preparation

The propagated bacteria were harvested by centrifugation for 15 min x 4500g at 4°C and plasmid DNA isolation was performed using QIAfilter Plasmid Midi Maxi Kits (Qiagen).

4.7.3.8 Sequencing

All plasmid DNA constructs were sequenced by MWG Biotech AG, in the

„Value read“ mode. The BCM Search launcher program was used for multiple sequence alignments.