6 replies to this topic

[gungrave]

Hello everyone, I have been running a couple of pcr samples on a 2% gel, what I notice is that I get my expected products and also a shadow band. What's even stranger is that my ladder also has a shadow band.
Has anyone experienced anything like this? I'm not sure if this is an annealing temp. problem due to the ladder. Thanks in advance.

[methylnick]

Hi Gungrave,

Is your agarose gel fresh? Another problem could be the size of the well, I have seen with a thick well I get shadows more often.

Nick

[gungrave]

Thanks for the reply. I do pour the gels fresh. The size of the wells sounds like a good suggestion. One of the problems is that this does not consistently happen. I will try switching combs. Thanks a lot.

[badcell]

Hi gungrave. Check also that the wells are not tilted. It happened to me once and I only noticed what was happening when I looked at the side of the gel- the comb had moved during gel preparation and the upper part of the wells was some mm in front of the lower part, so my bands were running tilted and when looking at them from above, it looked like they had a shadow.
Wow, that was difficult to explain. Hope you understand what I mean

[gungrave]

Wow, never thought of that. Thanks a lot   .
(P.S you did a good job explaining )

[fred_33]

hi
i know that a 2% gel is difficult to solubilize (don't know if it's the rght verb... ) make sure your solution is completely dissolved and let dry a longer time.

in the begining of my experiments, i didn't heat enough and it remained part of agarose not solubilized. That provoked shadowing.

Hem, hope i was clear too...

[gungrave]

Thanks, thats also a great suggestion.