bands position in agarose gel
Posted 29 December 2012 - 12:48 AM
we perform plasmid extraction using miniprep kit (alkaline lysis method).
Attached please find the gel. All the samples are of the same clone (lane 5 is hind III marker) but analysis perfomed on different time. Our results should look like the one in lane 1, however we get bands in higher mol wt. Can someone explain this and what we can do to overcome this.
we use TAE buffer during the run and add ethidium bromide during the agarose gel preparation only.
thanks in anticipation.
Posted 29 December 2012 - 08:15 PM
Also moved to the homework section.
Posted 31 December 2012 - 10:31 PM
Posted 01 January 2013 - 12:08 AM
The forms are generated both in the bacteria and by the extraction (2nd step is alkaline lysis) where the DNA is actually degraded by the alkalinity of the solution. The amount of relaxed circular is also dependent on the removal of protein during the extraction process.
Posted 02 January 2013 - 10:41 PM
If your miniprep failed to remove proteins effectively, you might be clogging your lanes and so your sample is getting into the lane late. If you look at the well up top, you can see there is some build-up right where the lane starts.
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