Concepciones de lectura en la contemporaneidad

3.3.1.2.1 Genomic DNA islolation from EDTA blood

For Southern blot analysis, genomic DNA extracted from EDTA blood (5 ml) using the Blood and Cell Culture DNA Midi kit® (Qiagen, Hilden, Germany) according to the manufacturer`s instructions was applied.

3.3.1.2.2 Restriction digest

Genomic DNA strands were cut into smaller fragments using a restriction enzyme. Besides of the DNA (8 µg) the reaction batch with a total volume of 30 µl contained 30 U of the restriction enzyme ApaI, its appropriate buffer as well as Spermidine (0.1 M). After an overnight digestion at 37°C another 10 U of the restriction enzyme were added to each sample followed by an incubation step of one hour. Prior to the use of the samples for gel electrophoresis 6 x loading dye was pipetted to each sample for a better monitoring of the gel electrophoresis process.

3.3.1.2.3 Gel electrophoresis and transfer of genomic DNA

DNA fragments were loaded into the gel slots of a 0.9% TAE agarose gel to separate them by size. A 1 kb ladder served as DNA molecular weight standard. When the samples, visualized by the bromophenol blue containing 6 x loading dye, reached the end of the gel, the gel electrophoresis could be stopped. The electrophoresis result was documented under UV-light using a gel documentation system. Prior to the blotting process, the gel was pivoted in 0.25 M hydrochloric acid for about 20 minutes depending on the color shift of the blue bands to yellow. After washing the gel for several minutes with aqua bidest. it was gently shook in denaturation solution until the color of the bands turned back to blue (around 45 minutes). Subsequently, the DNA was blotted onto a positive loaded nitrocellulose membrane by capillary transfer. Thereto the gel was placed upside-down on a plastic surface. Two layers of Whatman paper followed by multiple layers of absorbent paper were placed on the gel and weighed down by a weight of ca. 800 g to improve capillary transfer. The transfer was carried out overnight for 18-24 hours. The next day, the membrane was pivoted in neutralization solution for 5 minutes followed by 15 minutes of

air-drying. Cross linking of the transferred DNA with the membrane was accomplished under UV-light (120 J/cm2) before the membrane was stored at room temperature until further processing.

3.3.1.2.4 Probe establishment

The primers RIP2-sense and RIP2-antisense (see 3.3.1.1.2) were used to create a 720-bp probe. Plasmid DNA containing the RIP2-hGIPRdn construct in a concentration of 7 ng/µl served as template for the PCR.

Table 3.4 Reaction batch probe Southern blot PCR

Probe Southern blot 10 x Herculase buffer 10 µl

dNTPs (10 mM) 2 µl Sense primer (100 µM) 0.5 µl Antisense primer (100 µM) 0.5 µl Aqua bidest. 83.5 µl Herculase Taq Polymerase 0.5 µl

Template 3 µl

Total volume 100 µl

Table 3.5 PCR conditions probe Southern blot PCR

Probe Southern blot Denaturation 94°C 4 min Denaturation 94°C 1 min

Annealing 62°C 1 min 34 x Elongation 72°C 3.03 min

Final elongation 72°C 10 min

The amplified PCR product was separated in a 1% TAE-agarose gel after addition of 20 µl 6x loading dye. Then, the designated band was excised using a scalpel blade and DNA was extracted from the gel using the Jetquick Gel Extraction Spin Kit according to the manufacturer’s instructions. Subsequently, 2 µl of each extracted DNA sample as well as of the molecular weight standard Lambda/HindIII + EcoRI were loaded on a 1% TAE agarose gel. DNA concentration of the amplificates was estimated by comparison of the respective

band intensity with the intensity of the bands of the concentration standard Lambda/HindIII + EcoRI.

3.3.1.2.5 Radioactive labeling of the probe

The Rediprime II Random Prime Labeling System® was used to label 50-70 ng of the probe radioactively with 50 µCi α-[32P]-dCTP (GE Healthcare) according to the manufacturer’s instructions. Random priming is a method to label DNA. Oligonucleotides of accidential sequence are added to denatured single strand DNA for hybridization. The so called Klenow enzyme, a fragment of the DNA Polymerase I of E. coli, synthesizes the opposite strand using the oligonucleotides as primers. DNA is labeled by integration of radioactive nucleotides. A MicroSpinTM

S-300 HR column was used for removing all not incorporated nucleotides. Nuclear radiation per minute (cpm: counts per minute) was measured by a scintillation counter using 5 µl of a 1:100 dilution of the labeled probe. To calculate the cpm value per µl of the labeled probe the following formula, where Cerenkov is a correction factor for the calculation without scintillation liquid, was utilized:

cpm/µl = cpm * 20 (dilution) * 1.55 (Cerenkov)

3.3.1.2.6 Hybridization, washing and signal detection

Prehybridization of the nylon membrane preceded the hybridization step. Therefore the nylon membrane was incubated in Rapid-Hyb buffer® for two hours at 65°C in a hybridization oven. For hybridization, the labeled probe was used in a concentration of 2 x 106 cpm per µl Rapid-Hyb buffer®. After denaturation of the adequate amount of probe for five minutes at 95°C and chilling on ice, the sample was added to the prehybridization solution and incubated overnight at 65°C. The next day, nonspecific bound radioactivity was removed by three washing steps: 1 x washing solution I for 20 minutes (RT); 2 x washing solution II for 20 minutes (65°C). Blots were exposed in a Phosphor-Imager cassette and visualized with a Phosphor-Imager (Storm 860).

3.3.2 Physiological characterization of GIPRdn transgenic pigs