I have purified DNA of around 150 b.p whose sequence is similar to my gene of interest. Whenever I do normal PCR using common primers I get the band. While I have designed primers to later use for probe making for whole mount in situ hybridization by adding SP6 and T7 promoter but I am not getting the band instead I get smeared band. How to deal with it?
I don't completely understand your question or what PCR reaction you are running, but the smears are the result of primer extensions (i.e, PCR products) that run beyond the 150 bases your are expecting. Often if the amount of starting template is very concentrated, you'll get results that look like this. If you are amplifying from plasmid, the primers might be extending past the reverse primer thus giving longer fragments. You'll only see this if, again, your starting template is a high concentration. You might try diluting your template (many logs) and try again. Also, check the length of the viral primers and the annealing temperatures, they may be too short or low.
I agree with David as primer concentration may be high so try to reduce the primer concentration ,and by increasing the extension time and annealing temp u will increase the specificity .It will help to stop non specific binding by using powerful thermostable polymerase
I would increase the hybridization temperature, maybe reduce the time of elongation (for 150bp you probably do not need this step: elongaztion will occur during the increase and decrease of temperatures between cycles) have a look at your sp6/T7 primers for terminal nucleotides complementation...
I totally agree with Didier Poncet. Increase hybridization temperature and set the elongation time for 10 seconds.
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