Problem with agarose gel? or with RNA ? - (Nov/23/2006 )

Hiii

I am working on tuberculosis and right now I am extracting RNA from the fresh tissues.

I am facing problem with the gels that I am loading after RNA purification....

More than half of the sample is getting struck in the wells and its not running down..And my Gel is 1% agarose...At first I thought those are impurities getting struked in the gel. But all the gels are coming in the same way. My spectrophotometer values of RNA extarcted are good enough. So I really confused !!!
I am getting very lite bands but the sample got struck in the well is illuminating more than the bands I got..

can any one please help me with this question?

Thanks in advance

Hima

-hima-

QUOTE (hima @ Nov 23 2006, 05:46 PM)

It could be genomic DNA that you are carrying over. I would suggest you to run an old RNA sample that was clean or a plasmid at the side of your samples, just to be sure that you have it only in the RNA samples. But I'm pretty sure it's genomic. How are you extracting RNA?

-dnafactory-

how much are you loading?
if you load too much, it will get stuck in the wells.

V

-vetticus3-

I got this problem before.

After boiling the RNA with the LB, put them on ice for very short time to cool them down.
DO NOT put the RNA with LB on ice for too long, they become jelly-like and stuck in the well when running the agarose gel.

Hope this help.

-Minnie Mouse-

Is your RNA clean? Is it free of genomic DNA, proteins and polysaccarides. Got to make sure.

Next, are you drying your RNA? If you do, over drying can cause the RNA not to resuspend in solution. So the fine RNA percipitate will get retained within the well.

-perneseblue-

QUOTE (perneseblue @ Nov 23 2006, 05:51 PM)

Yes probabaly Iam doing that.... Thnaks a lot for helping me out...

Regards

Hima

-hima-

I am extracting using QUAIGEN kit method...

-hima-

Thank you for helping me with good answers. I will check with your suggestions and get back to you...

Regards

Hima

-hima-