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Position: Discussion forums > Bioscience biotechniques discussion forums > Molecular biology troubleshooting forum
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Backgrounds on blot
Posted by: Tang (IP Hidden, Unregistered user, )
Date: April 15, 2005 08:03AM
Our lab used to radioactive method for southern blot and I'm now testing the DIG kit from Roche.
And there is spotty background on the blot. I've used the Nylon membranes, hybridization bags and Hyb solution recommended from Roche. And UV X-link the blot instead of baking after transfer in 20XSSC. The blot is kept wet through the procedure from pre-hybridization to detection (drying of membrane shouldn't be the cause for background), all procedures follows the Roche protocol. Please could anyone help me with the problem and any tips for using Roche DIG detection? Also, the length of the bands seems to much shorten (the smallest band at only to half of the film) than detection with radioactive (the samllest band at near edge of film) and I've to run the electrophroesis for longer time and use 0.6% gel instead of 0.75% to get the bands run longer. Is there any reason for this?
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