It is not difficult to find researchers who swear by GITC- phenol procedures because good-quality RNA, free from geno- mic dNa contamination is quickly produced. However, a se- cond camp of researchers avoid these same procedures because they often contain contaminating genomic DNA (Lewis, 1997 S. Herzer, personal communication). There is no single expla nation for these polarized opinions, but the following should be considered Problems can occur in the procedure during the phenol/chloro form extraction step. The mixture must be spun with sufficient force to ensure adequate separation of the organic and aqueous layers; this will depend on the rotor as can be seen in Table 8.1 For best results the centrifuge brake should not be applied, nor should it be applied to gentler settings The interface between the aqueous and organic layers is another potential source of genomic contamination. To get higl purity RNA, avoid the white interface(can also appear cream colored or brownish) between the two layers; leave some of the aqueous layer with the organic layer. If RNA yield is crucial, you'll probably want as much of the aqueous layer as possible e. again leaving the white interface. In either case you can repeat the organic extraction until no white interface is seen. Residual salt from the precipitation step, appearing as a huge white pellet, can interfere with subsequent reactions. Excessiv salt should be suspected when a very large white pellet is obtained from an RNA precipitation. Excess salt can be removed br washing the RNa pellet with 70% EtoH(ACS grade). To the RNA pellet, add about 0.3 ml of room temperature (or -20oC 70% ethanol per 1.5ml tube or approximately 2 to 3 ml per 15 to 40ml tube Vortex the tube for 30 seconds to several minutes to dislodge the pellet and wash it thoroughly. Recover the RNA with a low speed spin, (approximately 3000 x g; approximately SS34 rotor), for 5 to 10 minutes at room temperature or ayos 7500rpm in a microcentrifuge, or approximately 5500rpm in Table 8. Spin Requirements for Phenol Chloroform Extractions Tube Speed Spin Time 1.5ml 10,000×g 2.0ml 15 ml 12,000×g 50ml 12,000×g RNA Purification 205It is not difficult to find researchers who swear by GITC— phenol procedures because good-quality RNA, free from geno￾mic DNA contamination is quickly produced. However, a se￾cond camp of researchers avoid these same procedures because they often contain contaminating genomic DNA (Lewis, 1997; S. Herzer, personal communication). There is no single expla￾nation for these polarized opinions, but the following should be considered. Problems can occur in the procedure during the phenol/chloro￾form extraction step. The mixture must be spun with sufficient force to ensure adequate separation of the organic and aqueous layers; this will depend on the rotor as can be seen in Table 8.1. For best results the centrifuge brake should not be applied, nor should it be applied to gentler settings. The interface between the aqueous and organic layers is another potential source of genomic contamination. To get high￾purity RNA, avoid the white interface (can also appear cream colored or brownish) between the two layers; leave some of the aqueous layer with the organic layer. If RNA yield is crucial, you’ll probably want as much of the aqueous layer as possible, again leaving the white interface. In either case you can repeat the organic extraction until no white interface is seen. Residual salt from the precipitation step, appearing as a huge white pellet, can interfere with subsequent reactions. Excessive salt should be suspected when a very large white pellet is obtained from an RNA precipitation. Excess salt can be removed by washing the RNA pellet with 70% EtOH (ACS grade). To the RNA pellet, add about 0.3ml of room temperature (or -20°C) 70% ethanol per 1.5ml tube or approximately 2 to 3ml per 15 to 40 ml tube. Vortex the tube for 30 seconds to several minutes to dislodge the pellet and wash it thoroughly. Recover the RNA with a low speed spin, (approximately 3000 ¥ g; approximately 7500 rpm in a microcentrifuge, or approximately 5500 rpm in a SS34 rotor), for 5 to 10 minutes at room temperature or at 4°C. RNA Purification 205 Table 8.1 Spin Requirements for Phenol Chloroform Extractions Volume Tube Speed Spin Time 1.5 ml 10,000 ¥ g 5 minutes 2.0 ml 12,000 ¥ g 5 minutes 15 ml 12,000¥ g 15 minutes 50 ml 12,000¥ g 15 minutes