Protocol for DNA Sequencing via PCR Clean PCR product 1. Add 100 μl PCR products to a 1.5ml microcentrifuge tube. Add 1000 μl Resi n and mix by vortex 3 minutes. 2. Take one disposable syringe, remove the plunger and plug one column (for DN A purification) on the tip of syringe. Transfer the PCR product and Resin mixt ure to the barrel of the syringe. 3. Insert the plunger to the syringe, plunge the solution through the column a nd discard the flow-through. 4. Detach the syringe from the column; remove the plunger from the syringe. At tach the syringe barrel back to the column. Add 2ml 80% isopropanol to the syr inge barrel. 5. Plunge the solution through the column and discard the flow-through. 6. Place the column on a 1.5ml microcentrifuge tube, cut the cap of the tube a nd spin at 13,000 rpm for 1 minute to remove residual isopropanol. 7. Place the column on a clean 1.5ml microcentrifuge tube. Add 50 μl distille d H2O; tap the tube slightly so that the membrane in the column can be fully c overed by H2O. Spin at 13,000 rpm for 1 minute to wash the DNA off. Quantify PCR Product 1. Take 5 μl clean PCR products, add 95 μl distilled H2O, and mix well. 2. Quantify DNA using distilled H2O as blank. Sequencing PCR 1. Set up two PCR sequencing reactions using one upstream primer and one downs tream primer (Sequencing from both direction), according to the following reci pe: Big Dye mix 4 μl Halfterm* 4 μl Clean PCR product 60 ng Primer 3.2 pmol d H2O 7.3 μl Total: 20 μl * Note: dNTPs, Taq polymerase, MgCl2, buffer included in the mix. 2. Run the PCR reaction in a thermal cycler as the following conditions: 25 cy cles of (1.denaturation at 96°C for 5 minutes; 2. annealing at 50°C for 5 mi nutes; 3. extension at 60°C for 5 minutes.) Precipitate Sequencing Runs 1. Add 2 μl 3 M NaOAc, pH 4.6, 50 μl 95% EtOH and 20 μl sequencing runs to a 1.5 ml microcentrifuge tube. 2. Vortex and let stand for 15 minutes. 3. After step 2, centrifuge the tube at highest speed for 20 minutes. Carefull y remove the supernatant. 4. Wash the pellet with 250 μl 70% ethanol, spin at highest speed for 5 minut es. 5. Remove the supernatant by pipetting. Set the tube at room temperature to re move the remaining EtOH. Submit the sequences to Genebank Go to the NCBI homepage (http://www.ncbi.nlm.nih.gov/), run a standard nucleot ide BLAST search.If not working, try translating the sequence into a.a seq. th en run a peptide seq. BLAST. (责任编辑:泉水) |