I digested bacterial plasmid with EcoRI and EcoRV. digested product was smear. But the plasmid has only two RE site.
The smear is due to non specific nuclease contamination. It may come from your buffer, your restriction enzyme, or your DNA prep. I would suggest making fresh buffer from autoclaved solutions and make sure that your pipette tips are autoclaved. Also, avoid digesting your sample longer or with more enzyme than what is actually needed
It may also because of star activity of enzymes which cut the sequence in multiple bands.
I agree with Pierre. you can check nuclease contamination simply NOT adding your enzymes in the mixture and do use the same reagents.
how many times did you repeat the digestion?
If I assume it is not a contamination, then next time when you perform, use two sets one for partial digestion and other complete digestion and see if u get smearing. share the result., see the band pattern
and yes use fresh reagents and autoclaved tips, wear gloves to avoid nuclease contamination.
check that you do not have too much ecorI as this has star activity and can cut in many places as Saman says if there is too much glycerol present. Try also cutting with each enzyme first and checking the cutting then use the other enzyme rather than a double digest in one reaction. The results may be informative as to where the problem lies. Does the uncut plasmid also have this smear in which case perhaps it is RNA and you just do not have much DNA. Also bacterial dna will cut into a smear if there is high molecular weight dna present in which case you will need to check your plasmid preparation is working properly
Paul is right.
Try and use lesser units of enzymes in 20ul reaction (0.2-0. 3ul) and perform reaction for longer time 45-60 min.
Secondly you can also try and heat denature your enzymes before loading.
Thirdly, you can perform 2 single digestions...... first with ecor1 followed by ecorv.... with purification step in between
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