Streaky DNA digest - (Apr/02/2010 )
i am trying to digest a plasmid to use as my vector but when i digest it the bank comes out streaky. it looks similar to this (lane 1).
This is not my gel but just some random picture i found on google but that's how my band looked. i've had vectors digest like this before and they were fine but this time the concentrations of my gel extractions were unusually high at 100-260ng/ul. what do you think is going on? i've digested this plasmid with other enzymes and got crisp bands. could it be contamination? and if so, would a PCR purification with a qiagen kit help solve the problem? or have any of you had this happen before (unusually high concentration of fragment after gel extraction) and they still worked fine?
do you mean the like streaks present in every lane in the attached picture, or the blurriness of band1
if it is the blurriness; normally I would say that the well have been overloaded with DNA. And that is the cause of your problem. The width of DNA bands in this gel (pic) suggest a hefty amount of DNA in the wells.
However, if digest with other enzymes give sharper bands (assuming the same amount of DNA is loaded) it could be cause by having a little more salt in the sample coming from the buffer. This suggestion works if your blurred DNA band was digested in a high salt buffer and the nice sharp one was digested in a low salt buffer.
If you mean the light streak; it is indication of genomic DNA contamination.
Binding to a silicon column will not help. Gel purification would help remove most of the gDNA contamination. The blurriness of the bands can be helped by loading less DNA into the well.
thanks for the advice. yea my gel is like band 1. it definitely had too much dna. i was trying to digest about 14ug of DNA because of a mis-communication of the plasmids concentration.
just trying to figure out what else is occurring here so, why is it that my fragment concentration is so high after gel purification? is it probably due to the fact that i had just too much DNA to digest so its not all digested DNA?
i tried my digest again with half as much DNA but my bands still came out streaky and the concentrations are still high. normally i wouldn't worry about it but when i test the concentration, the graph of absorption vs wavelength doesn't yield just one peak but instead it shows about 2 peaks and one small bump. What could be causing these other peaks? could it be undigested plasmid? I don't think there is contamination because i digested this plasmid before but purified the fragment instead of the vector and it gave me only one single peak.