need help with PCR gel

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need help with PCR gel

Post by biosis123 » Tue Feb 07, 2012 2:57 am

I made a PCR product of 20ul total, ran 3ul of the product on a 2% agarose pre-cast gel. This is the first picture. There is a nice band at the correct bp size (400bp), but a fainter band is above it. So I wanted to gel purify out my correct band.

I ran the 17ul leftover on a poured 2.5% TBE gel, at 100V for 40minutes. The DNA ladder on the right lane is a 100bp ladder, so my product is 4 bands from the bottom, at 400bp. But there is a large smear running down and this gel looks nothing like that of the pre-cast gel. I am afraid I cannot purify this band because I lost some of the product due to smearing.

What does this smear mean? Did I overload the gel, did I run gel too fast? Should I use TAE instead of TBE? I always run with those conditions, so I didn't do anything out of my norm. But I need to purify only that band seen in the precast gel, how can I make it look the same on the poured gel?

I appreciate any advice. Thanks
second gel
gelpic2.jpg (10.72 KiB) Viewed 1472 times
first gel
gelpic1.jpg (9.08 KiB) Viewed 1472 times

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