I did a colony pcr reaction (final vol 100ul) and the product was electrophoresed in 1.2% agarose gel . After complete run I observed a smear from one end to another end in each well (of the gel) including blank (without template).
I thought that problem with concentration of DNA template and tried in other trials by reducing the concentration of the template. But I got the same result.
To overcome the contamination problem I tried with new components, new plastic ware and auticlaved glass ware
but the result is same.
Can anybody suggest me to overcome this problem?
This is the set up I performed
10X Taq Buffer-10ul
10mM dNTPS -2 ul
M13R Primer (3pmol?ul)-2ul
Template-usually 4 ul
You’ve changed everything, but it’s still in your blank? Then it suggests your changed stuff is still contaminated…find a lab with working results and see if they will let you borrow their stuff. If that still doesn’t work, then it sounds like your protocol is contaminating it.