In order to do cloning, we had done double digestion of plasmid with SphI and KpnI restriction enzymes; but after electrophoresis, we did not see any band on the gel. The enzymes are produced by Bioron Company and we used KpnI buffer for double digestion. Our digestion condition is:
(µl)
µl 15 plasmid
µl 3 buffer (10X)
µl 3 BSA (1 mg/ml)
µl 0.5 Kpn I (10 u/µl)
µl 0.5 Sph I (10 u/µl)
µl 8 DDW
µl 30 Final volume
2 hrs---- 37 'C
Dear Mostafa, If you cannot even see you plasmid DNA on the gel, it strongly suggests that you have a problem with the isolation of the plasmid DNA in the first place, or its storage before use. The easiest way to sort this out is to make another preparation of plasmid DNA (using a miniprep kit if possible), and to digest aliquots of this with no enzyme (the negative control) and enzymes of interest one at a time, and then any double-digest you are interested in. You should then run this on a gel and look to see if the DNA is present, degraded, or restricted as expected. If there is sufficient Mg2+ in your DNA preparation (as is often the case with fast DNA preps), you will have DNAse activity slowly destroying your plasmid DNA. This also happens when you add RE buffers (as they are also a source of Mg2+) during the digestion - you can loose all of your DNA in 30 - 60 min and will see nothing on a gel. Regards, Andrew
It could be because the two enzymes you chose are incompatible. In fact, the optimal buffer's compositions for each enzyme of that company are quite different ( http://www.bioron.net/en/products/restriction-endonucleases/h-r/kpn-i/ ) vs ( http://www.bioron.net/en/products/restriction-endonucleases/s-z/sph-i/ ). If you really need to cut with these 2 enzymes, personnally I would use NEB's, as they provide more information on buffer compatibility. For example, they say that KpnI and SphI would be compatible (100% efficiency of both) using buffer 1.1 ( https://www.neb.com/tools-and-resources/usage-guidelines/nebuffer-performance-chart-with-restriction-enzymes ). More info also here https://www.neb.com/tools-and-resources/usage-guidelines/double-digests
KpnI is fickle. In my experience, the enzyme goes bad very quickly, even if kept cold all the time.
I also agree with Jean regarding the NEB enzymes.
An easier answer would be the amount of plasmid (ug) you are puting in the rxn.
If the fragments are small then they won't be visible in the gel. Try with at least 1ug.
It isn't clear, did you see nothing on your gel or just not the expected band but you did see the vector? The possible problems depends upon what you observed.
Thanks all
Dear Michael
we cannot see anything, Even we cannot see our plasmid!
If you don't see the plasmid on the top of your gel then I think it just means you are not putting enough plasmid DNA in your rxn.
Dear Mostafa, If you cannot even see you plasmid DNA on the gel, it strongly suggests that you have a problem with the isolation of the plasmid DNA in the first place, or its storage before use. The easiest way to sort this out is to make another preparation of plasmid DNA (using a miniprep kit if possible), and to digest aliquots of this with no enzyme (the negative control) and enzymes of interest one at a time, and then any double-digest you are interested in. You should then run this on a gel and look to see if the DNA is present, degraded, or restricted as expected. If there is sufficient Mg2+ in your DNA preparation (as is often the case with fast DNA preps), you will have DNAse activity slowly destroying your plasmid DNA. This also happens when you add RE buffers (as they are also a source of Mg2+) during the digestion - you can loose all of your DNA in 30 - 60 min and will see nothing on a gel. Regards, Andrew
Dear Mostafa
First, check the DNA concentration by gel elctrophoresis & spec.
In case of double digest, 1ug plasmid would be necessary
You can also do the digest separately if there's problem with incompaible buffer.
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