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Troubleshooting Guide for End-Point PCR Problem No product or low yield Non specific products Product in negative control Possible Solution Make sure you added all reaction components (e.g. template DNA, primers, buffers, MgCl2 etc.). Make sure there are no inhibitors in DNA sample (e.g. heparin) Double check the sensitivity and compatibility of your primers. Verify that there are no primer-dimers. Try increasing MgCl2 concentration. Increase primer amount. Increase template amount. Verify template purity and integrity. Change the dNTP solution. ® If using HOT FIREPol , make sure you wait for the activation time (12 to 15 minutes initial heating). Gradually increase extension time. Gradually decrease annealing temperature. Add enhancing agents e.g. DMSO, PEG, BSA, Solution S. Add increments of MgCl2. Check that the wells on the gel are loaded correctly and that the loading buffers and ethidium bromide are added If using normal PCR, try using Hot Start instead. Lower MgCl2 concentration. Try all the buffers provided. Lower template concentration. Lower annealing temperature gradually. Decrease annealing time. Lower primer concentration. Increase extension time. Make sure you do not have contamination from impure template or reagents. Always use filter tips. Wear gloves. Avoid leaving tubes lids open to avoid aerosol contamination. Solis BioDyne Riia 185a, 51014 Tartu, Estonia, tel: +372 740 9960, fax: +372 740 2079, e-mail: [email protected], www.sbd.ee