DNA extraction protocols

1.5.1 Extraction of total mouse/hamster genomic DNA

Solutions used:

Solution 1 (w/v): Solution 2 (w/v) 50 mM T ris. 01® (pH7.5) 2.6 M NaCI 400 mM NaCI®

100 mM EDTA® ( Ethylenediaminetetraacetic Acid) 0.5% SDS (Sodium Dodecyl Sulfate)

0.01% Proteinase K

Mouse genomic DNA was extracted from the lungs, spleen and liver of mice. The tissue was teased apart with sterile scalpel blades and placed in solution 1, in a 30 ml polypropylene tube. For lungs and spleens I used 5 ml and for livers 8 ml of solution 1. The cap of the tubes was perforated with a 19 g needle to stop the pressure building up from gases released during the digestion. The digestion was carried out at 55°C with constant shaking (100 rpm) for 24-48 hours. If the digestion was not complete after 24 hours, the tubes were vortexed for 5 seconds to break up partially digested tissue and the incubation continued for an additional 24 hours. After the digestion, the concentration of the NaCI was brought to 1.5 M by adding an equal volume of solution 2, the tube shaken for 15 minutes, followed by centrifugation at 10000 g for 5 minutes. The supernatant was collected into a clean tube and the DNA precipitated by the addition of one volume of ethanol. The DNA was spooled out, dissolved in ddH20 and stored at 4°C.

The hamster DNA was extracted from total hamster tissue. The animals were killed by cervical dislocation and their skin and digestive tract removed. The remainder of the tissue was placed in liquid Nitrogen for about 20 seconds (until the effervescence stopped) and the frozen tissue ground in a blender. One gram of powderised tissue was digested in 5 ml of solution 1 for 24 hours and the DNA precipitated as described above. Because the DNA in the tissue sample was extensively sheared during the blending process, the DNA was precipitated at -70°C for 20 minutes, collected by centrifugation at 1000 g for 15 minutes at 4°C and resuspended in ddH20. This DNA was used only for competing out repetitive DNA sequences.

1.5.2 Extraction of total Yeast DNA

Solutions used: Solution 1: 40 mM 90 mM EDTA 2-mercaptoethanol Solution 3: 50 mM 25 mM 500 mM 3 mM 1 % (w/v) Tris. Cl (pH7.4) EDTA NaCI 2-mercaptoethanol SDS Solution 2 (w/v): 1 M Sorbitol 60 mM EDTA 0.1 M Sodium citrate pH adjusted to 7.0, and 2 mercaptoethanol (BDH; 14.25M) added at 1:125 dilution as well as 1 mg/ml of Lyticase (Sigma)

Solution 4:

Phenol:chloroform:isoamyl alcohol at the ratio of (1:1:0.02)

Cells from a 20 ml culture were harvested by centrifugation at 2000g for 10 minutes, washed twice in solution 1, resuspended in 2 ml of solution 2 and incubated at 37°C stationary in a 10 ml polypropylene tube for 3 hours. Because yeast cells tend to sediment very quickly when stationary, the tube was laid on its side to spread the cells along its entire length, thus maximising the effectiveness of the Lyticase action. After the Lyticase treatment, the spheroplasts (yeast cells stripped from their cell wall) were centrifuged at 3000g for 10 minutes, resuspended in 0.5 ml of solution 3 and incubated at 65°C for 15 minutes with intermittent vortexing. This was followed by two extractions with solution 4, one extraction with chloroform and precipitation of the DNA with one volume of isopropanol at room temperature for 15 minutes. The DNA was collected by centrifugation in a microfuge at 13000 g for 15 minutes, washed with 1 ml of 70% ethanol and the pellet dried by heating the tube at 75°C for about 2 minutes. The DNA was then resuspended in 300 pi of TE (pH 8.0) and incubated for 4 hours at 37°C in the presence of 2 pg of DNase-free RNase (prepared by boiling RNAase in 15 mM NaCI and allowed to cool to room temperature; stored at -20°C). The DNA was

again precipitated with one volume of isopropanol, washed with 70% ethanol and resuspended in 200 |il of TE. From a 20 ml culture about 400 pg of DNA was obtained.

1.5.3 Extraction of plasmid and bacteriophage X DNA

Plasmid DNA was extracted using a procedure based on alkaline lysis of bacterial cells and the reagents used are commercially available (Promega; Magic Minipreps™ Purification). The protocol followed was as recomended by the supplier. DNA prepared in this way was used for all the applications that are described elsewhere in this thesis. Bacteriophage X DNA was also extracted using a commercially available reagent called LambdaSorb Phage Adsorbent (Promega).

1.5.4 Immobilisation of high molecular weight DNA in agarose blocks for PFGE 1.5.4.1 Mouse DNA

Solutions used:

Phosphate-buffered saline (PBS)* (w/v): Solution 1 (w/v):

30 mM NaCI 250 mM EDTA*

2 mM KOI (Potasium Chloride) 1% Sarkosyl* 10 mM N82HP04 (di-Sodium Hydrogen 0.4% Proteinase K

Phosphate)

1 mM KH2PO4 (Potasium di-hydrogen Phosphate)

pH adjusted to 7.4.

1% ‘UltraPure’ low melting point (LMP) agarose held at 42°C.

Freshly prepared 0.004% Phenylmethylsulfonylfluoride (PMSF), dissolved in isopropanol.

High molecular weight DNA was prepared from the thymus of a 21-day old female mouse, strain CSH/HeH. Mice were killed by cervical dislocation in a designated area. The thymus was removed and placed in 10 ml of PBS. To release the thymocytes, the thymus was squeezed between two scalpel blades. After carefully removing the connective tissue, the cells were collected by centrifugation at 1100 g for 10 minutes and the pellet resuspended in 6 ml of PBS. This volume was determined empirically and was always used, provided a 21-day old C3H/HeH mouse was used. The cell suspension was subsequently mixed with 6 ml of molten agarose, mixed thoroughly by gentle shaking, and dispensed into 100 pi block-formers (5 mm x 2 mm x10 mm). After the agarose hardened, the blocks were transferred into solution 1 (20 ml / 30 blocks) and incubated at 50°C for 48 hours with very gentle shaking (30 rpm). This was followed by 2 washes in ddH20 for 10 minutes at room temperature and three 30-minutes washes in TE (pH7.5) containing 0.04 mg/ml of PMSF. The blocks were stored in 0.5 M EDTA at 4°C. For PFGE, 1/2 or 1/3 of a block contained adequate

DNA (i.e. about 10 pg) for analysis by Southern blotting.

1.5.4.2 Yeast DNA Solutions used:

1 M 0.1 M 10 mM EDTA Sorbitol sodium citrate, pH 5.8 10 mM 50 mM Tris .Cl (pH 7.5) EDTA (pH 7.5 Solution 2: 400 mM EDTA' 1 % Sarkosyl 0.4% Proteinase K Solution 3:

SCE plus 10 mM Dithiothreitol (DTT^; prepared in 0.1 M sodium acetate, pH 5.2)

Solution 4:

1.5% of freshly prepared LMP agarose in SCE containing 1 mg/ml of Lyticase, kept at 45°C. The cells from a 20 ml culture were collected by centrifugation at 2000 g for 10 minutes, resuspended in 10 ml of solution 1, collected again by centrifugation and resuspended in 10 ml of SCE. After centrifugation, the cells were resuspended in 1 ml of SCE, mixed thoroughly with 1 ml of LMP agarose (as above) and dispensed into 100 p.1 block-formers. After the agarose hardened, the blocks (20 in total) were placed in 20 ml of solution 3 for 2 hours at 37°C with very gentle shaking (30 rpm). Finally the blocks were placed in 20 ml of solution 2 and incubated at 50°C for 48 hours again with gentle shaking. The subsequent steps were as in section 1.5.4.1.

1.5.5 Quantification of DNA

The concentration of the DNA in solution was determined spectrophotometrically, by measuring the optical (OD) density at wavelengths of 260 nm and 280 nm in a CIBA CORNING 2800 spectrascan. An OD260 of 1 corresponds to

50 pg/ml of double-stranded DNA. For pure DNA the ratio of (OD260/ OD280) was

between 1.8 and 2.0 (Sambrook et ai, 1989).