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Smear in my restriction digest - (May/07/2009 )

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If you use the QIAgen miniprep Kit with endA+ strain, remember to perform the recommended step:Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 3060 s.
Possibly the endonuclease is contaminated into your water, not buffer or your preps sample (but it needs the buffer with Mg2+ to be active). Did you change your water? I suggest using the sterilized water supplied with some enzymes which made by a company. If you suspect your loading buffer, just run the control (plasmid without digestion)


it sometimes happen that the enzyme have higher binding affinity to dna. so it appears like smear on agarose. you can try adding 0.1-0.5% SDS(of final reaction volume) to reaction mix before running gel. sds will denature the restricion enzyme.


It can have other reasons as well ...
Try to lower the voltage you run the gel at to below 130 volt. Some use 150 and especially in case of long fragments that means the gel has to run quite long at a high voltage and will become quite hot decreasing resolution quality.
In case your fragments (or plasmid) are bigger than 3kB make sure you use TAE instead of TBE as the resolution of TAE for long fragments is much better. But run TAE at 110V max, it has a lower buffer capacity and gets hotter earlier once more decreasing the resolution quality.

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