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Additional info for Bacterial, Phage and Molecular Genetics: An Experimental Course
05 M Na3EDTA and washed in distilled water. 56 7. Thermal De- and Renaturation of DNA from Different Organisms The DNA of all organisms and many viruses is, in its native state, a double-stranded molecule (see Expt. 6). The hydrogen bonds between the complementary bases A-T and G-C as well as the stacking forces between the neighboring bases are responsible for the double helical structure of DNA and its stability. , the DNA becomes single-stranded and the single strands form random coils. The lower the guanine and cytosine content of the DNA, the easier this denaturation occurs.
Immediately resuspend each pellet in 8 ml or even less of P-buffer by blowing the buffer with a pipette repeatedly over the pellet until it is resuspended. If more time is available, phage pellets may be overlayered with buffer and allowed to sit overnight to resuspend. Centrifuge the resuspension 10 min at low speed (SS34 rotor, approx. 4,500 rpm) and use only the supernatant. 2. Purification with CsCI. Fill about 24 ml of phage suspension obtained in step 1 into centrifuge tubes (No. 55 g/cc).
At low pH, the N-glycosyl linkages between the deoxyribose and the purine residues are split, resulting in an apurinic acid. The extraction of DNA from phages is quite easy, because in phages the ratio of protein to DNA is about 1:1, whereas in bacteria and other cells protein dominates. In addition, in phages there are no cellular nucleases nor polysaccharidic cell wall components to complicate the DNA extraction. S x 10 7 ). The T-even phages (T2, T4, T6) contain S-hydroxymethyl cytosine (HMC) instead of cytosine.