PEG PRECIPITATION OF PCR PRODUCTSThis protocol can be used instead of EXO/SAP for removing excess primers and nucleotides from PCR products before cycle-sequencing. Protocol: 1. After PCR, add the following reagents to each tube: For 30 µl reaction add: 4.8 µl of 5 M NaCl 4.8 µl of TE buffer 8.4 µl of 40% PEG-8000, 10mM MgCl2 For 50 µl reaction add: 8 µl of 5 M NaCl 8 µl of TE buffer 14 µl of 40% PEG-8000, 10mM MgCl2 2. . After adding the PEG, stir pipet in tube and pipet up and down to mix. Make sure all solution remaining in the tip is injected into the tube. 3. Incubate at room temperature for 15 minutes. Note: Pipet PEG solution slowly. PEG solution is viscous and may hold up in tip. If doing reactions in bulk, the three reagents may be mixed and then aliquoted in either 18µl or 30µl amounts per tube. 4. Pellet the amplification reaction products by centrifugation at 3250rpm for 30 minutes at 4oC. 5. Remove most of the supernatant by inverting the tray and carefully and gently decanting the supernatant. Remove remaining supernatant by inverting the tray, with paper towel underneath, and centrifuging at 300 rpm for 3 minutes. Repeat with clean paper towel. 6. Air dry the tubes for a few minutes and then resuspend each sample in 25 µl of water. Vortex to mix. Load 2 µl on a 0.7 % agarose gel to verify that DNA is present. Run at 55 Volts for 30 minutes. 7. Use 1 µl for dye-terminator cycle-sequencing reactions. If reactions are top-heavy, dilute by 1/2 and repeat sequencing reaction. |
