Remove the ethanol carefully, as the pellets may not adhere tightly to the tubes. The tubes should then be respun briefly and the residual ol removed by aspiration with a drawn out Pasteur pipet. Repeat this wash if the pellet seems unusually large Non-Phenol-Based methods Very fast, clean RNA, can process large sample numbers, pos ble genomic contamination One major drawback to using the guanidium acid-phenol nethod is the handling and disposal of phenol, a very hazardous chemical. As a result phenol-free methods, based on the ability of glass fiber filters to bind nucleic acids in the presence of chaotro- pic salts like guanidium, have gained favor. As with the other methods, the cells are first lysed in a guanidium- based buffer. The lysate is then diluted with an organic solvent such as ethanol or isopropanol and applied to a glass fiber filter or resin DNA and proteins are washed off, and the rna is eluted at the end in an aqueous buffe This technique yields total Rna of the same quality as he phenol-based methods. DNA contamination can be higher with this method than with phenol-based methods(Ambion, Inc unpublished observations). Since these are column-based proto- cols requiring no organic extractions, processing large sample numbers is fast and easy. This is also among the quickest methods for RNA isolation, usually completed in less than one hour The primary problem associated with this procedure is clogging of the glass fiber filter by thick lysates. This can be prevented by using a larger volume of lysis buffer initially. A second approach is to minimize the viscosity of the lysate by sonication(on ice, avoid power settings that generate frothing) or by drawing the lysate through an 18 gauge needle approximately 5 to 10 times. This step is more likely to be required for cells grown in culture than for lysates made from solid tissue. If you are working with a tissue that is known to be problematic (i.e, high in saccharides or fatty acids), an initial clarifying spin or extraction with an equal volume of chloroform can prevent filter-clogging problems A rea- sonable starting condition for the clarifying spin is 8 minutes at 7650xg. If a large centrifuge is not available, the lysate can be divided into microcentrifuge tubes and centrifuged at maximum speed for 5 to 10 minutes. Avoid initial clarifying spins on tissues rich in glycogen such as liver, or plants containing high molecular weight carbohydrates. If you generate a clogged filter, remove the remainder of the lysate using a pipettor, place it on top of a fresh filter, and continue with the isolation protocol using both filters. 206 Martin et alRemove the ethanol carefully, as the pellets may not adhere tightly to the tubes. The tubes should then be respun briefly and the residual ethanol removed by aspiration with a drawn out Pasteur pipet. Repeat this wash if the pellet seems unusually large. Non-Phenol-Based Methods Very fast, clean RNA, can process large sample numbers, possi￾ble genomic contamination. One major drawback to using the guanidium acid-phenol method is the handling and disposal of phenol, a very hazardous chemical. As a result phenol-free methods, based on the ability of glass fiber filters to bind nucleic acids in the presence of chaotro￾pic salts like guanidium, have gained favor. As with the other methods, the cells are first lysed in a guanidium-based buffer. The lysate is then diluted with an organic solvent such as ethanol or isopropanol and applied to a glass fiber filter or resin. DNA and proteins are washed off, and the RNA is eluted at the end in an aqueous buffer. This technique yields total RNA of the same quality as the phenol-based methods. DNA contamination can be higher with this method than with phenol-based methods (Ambion, Inc., unpublished observations). Since these are column-based proto￾cols requiring no organic extractions, processing large sample numbers is fast and easy. This is also among the quickest methods for RNA isolation, usually completed in less than one hour. The primary problem associated with this procedure is clogging of the glass fiber filter by thick lysates. This can be prevented by using a larger volume of lysis buffer initially. A second approach is to minimize the viscosity of the lysate by sonication (on ice, avoid power settings that generate frothing) or by drawing the lysate through an 18 gauge needle approximately 5 to 10 times. This step is more likely to be required for cells grown in culture than for lysates made from solid tissue. If you are working with a tissue that is known to be problematic (i.e., high in saccharides or fatty acids), an initial clarifying spin or extraction with an equal volume of chloroform can prevent filter-clogging problems. A rea￾sonable starting condition for the clarifying spin is 8 minutes at 7650 ¥ g. If a large centrifuge is not available, the lysate can be divided into microcentrifuge tubes and centrifuged at maximum speed for 5 to 10 minutes. Avoid initial clarifying spins on tissues rich in glycogen such as liver, or plants containing high molecular￾weight carbohydrates. If you generate a clogged filter, remove the remainder of the lysate using a pipettor, place it on top of a fresh filter, and continue with the isolation protocol using both filters. 206 Martin et al