Colony PCR

[shivakumar]

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
M13F primer(3pmol/ul)-2ul
M13R Primer (3pmol?ul)-2ul
Taq- 1ul
Template-usually 4 ul
Sterile water-79ul

PCR Programme
94c-5'
94c-1'
55c-1:30'
72c-1'
30 cycles
72c-10

[mith]

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.

[pcrboy]

determining where the smear appears may help determine whether it is chromosome DNA or plasmid DNA.