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smearing in the restriction digested products - (Sep/28/2007 )

hi everyone!

In order to clone a pcr product, i am using EcoRV and NotI to digest the plasmid and the topo clone containing my pcr product. After 1hr incubation of the 20ul reaction mix, on running the products on the gel i m getting a trailing smear below the digested product in case of the plasmid... Cud anybody please tell me the reason. unsure.gif In order to avoid the dnase contamination i have used everything fresh right from the buffer to BSA to sterile MQ. Smearing is also observed in the control reaction without enzymes....

Kindly help me out on this mellow.gif


Sounds like just a smear from the PCR reaction, which seems to happen sometimes. If you have a major band of the correct size with a bit of smearing, it's probably nothing to worry about.

Did you check the PCR product on a gel before the restriction digest; was it smeared then?
Could you post a photo of the gel?

-bitesizebio guy-

Did you run your undigested plasmid as a control? I mean, if you can get a nice band for the uncut plasmid, I am pretty sure it is star activity. Hope it helps.