Pulse Field Gel Electrophoresis - smear at lower molecular weight bands (Oct/14/2009 )

This is a PFGE of pseudomonas aeruginosa. There is a smear at the lower molecular weight bands..Wondering any advices I can get to troubleshoot this? I am thinking it could be DNA degradation?

This is the pulse field image for pseudomonas..






Lane 1,8 & 15 are my ladders....

Thanks!

What pulse reversal intervals are you using? The marker looks good, so that may not be the problem, but it can definitely blur bands if it isn't right. Is the buffer fresh? Growth of things in the buffer can be another problem.

The buffers were prepared about less than 2 months ago..

Were you able to figure out what to do to remove the smearing at the lower molecule weight bands? I have the same problem where my higher weight bands are clear but the lower weight ones smear. Is this related to my pulse times? Any suggestions?

Hello

I am not sure about the electrophoresis setting but if u follow the protocol reccomendation should be fine..are u working with pseudo? anyway it is strange but when I used a fresh gel stain it actually reduces the smearing and overall have clearer bands compared to using an old stain(roughly used about 5 times).
Perhaps u can try using a fresh gel stain when u do this and see if it reduces the smearing at lower mol wt. For me it helps using fresh stain but i am not sure why though!!! muahhaha I do try to cut my dna agarose plug smaller too.

I read that using thiourea during your electrophoresis can be helpful to reduce smearing, i didnt try but u can try that?

GoodLuck!!

mich

Hi,
Thanks for the response. Which protocol recommendation are you referring to? I am not working with pseudo but with fusobacterium, prevotella and porphyromonas. I have been using a fresh gel stain for all of gels so far, as well as thiourea, which has helped a bit but still not enough to get rid of the smearing.

ocean_sky83 on Feb 28 2010, 12:32 AM said:

Hello

I am not sure about the electrophoresis setting but if u follow the protocol reccomendation should be fine..are u working with pseudo? anyway it is strange but when I used a fresh gel stain it actually reduces the smearing and overall have clearer bands compared to using an old stain(roughly used about 5 times).
Perhaps u can try using a fresh gel stain when u do this and see if it reduces the smearing at lower mol wt. For me it helps using fresh stain but i am not sure why though!!! muahhaha I do try to cut my dna agarose plug smaller too.

I read that using thiourea during your electrophoresis can be helpful to reduce smearing, i didnt try but u can try that?

GoodLuck!!

mich

Hi

Are u using ethidium bromide stain for your PFGE?