Biology-Online • View topic - need help with PCR gel
Login

Join for Free!
121818 members
Advertisement
Advertisement

need help with PCR gel

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

Moderator: BioTeam

need help with PCR gel

Postby 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
Attachments
gelpic2.jpg
second gel
gelpic2.jpg (10.72 KiB) Viewed 906 times
gelpic1.jpg
first gel
gelpic1.jpg (9.08 KiB) Viewed 906 times
biosis123
Garter
Garter
 
Posts: 1
Joined: Tue Feb 07, 2012 2:54 am

Return to Molecular Biology

Who is online

Users browsing this forum: No registered users and 2 guests