Smeared PCR product - ISSR marker screening (Jan/15/2007 )
i did ask this question beofre but no replies. iam getting smeared PCR product when i am doing the reaction with ISSR primers purchased from UBC. i have changed the conc of DNA too. the calculated Tm for this primer which is 18 mer (CCG)6 is around 72 degrees. i tried the reacion with 61 degrees. could that be the problem? can i try at 70 or 68 degres? pl suggest a solution to this . also UBC states that most of their primers work at 55 degrees,
When you see a smear in a gel could be cause by:
excess of template (use always the least quatity possible and raise it if needed).
degrate DNA (always prepare a working solution and mantein a stock, so you will alway have DNA)
excess MgCl or enzyme
A low anneling temp causes lots of bands (unspecify products), so always compare the Tm of primer given by company (check the sheet that must sent with them) and the expected, if there is to much difference use a temp in between.
It will be better to see a photo, but I gave you the must general causes.hope this help!