PCR and Gel Electrophoresis troubleshooting
1) Try using different concentrations of template DNA (e.g., 50x, 100x, 200x) for initial trials to optimize the amount used. 2) Run unamplified DNA samples against the size ladder to ensure the smear isn't due to excessive template DNA. 3) Measure the OD260 of the sample and amplify 25 ng of DNA or use serial dilutions (e.g., 1:2, 1:4, 1:8, 1:16) to test the best concentration. 4) Lower the amount of primer and ensure the DNA sample is pure, as impurities can also cause smears. 5) Consider reducing the number of PCR cycles if the reaction is over-amplified. https://www.youtube.com/watch?v=Mwj5FTWcJ6c
1) Try using different concentrations of template DNA (e.g., 50x, 100x, 200x) for initial trials to optimize the amount used. 2) Run unamplified DNA samples against the size ladder to ensure the smear isn't due to excessive template DNA. 3) Measure the OD260 of the sample and amplify 25 ng of DNA or use serial dilutions (e.g., 1:2, 1:4, 1:8, 1:16) to test the best concentration. 4) Lower the amount of primer and ensure the DNA sample is pure, as impurities can also cause smears. 5) Consider reducing the number of PCR cycles if the reaction is over-amplified. https://www.youtube.com/watch?v=Mwj5FTWcJ6c