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Enzyme cutting: low efficiency


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5 replies to this topic

#1 DrLeo

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Posted 26 March 2013 - 05:55 PM

Hi all,
I used SacI and XhoI to cut out my inserts with the protocol as below:
plasmid DNA 10 ul
SacI 0.15 ul
XhoI 0.15 ul
10X M buffer 1.5 ul
D.Water 3.2 ul
----------------------
Total 15 ul

I incubated the reaction (in 1.5ml eppendorfs) at 37oC, 2h

After loading on gel for checking, the concentration of plasmid DNA was so high, but the concentration of the insert was low...
How can I do to increase the efficiency of this reaction? I need high concentration of insert for ligation step.

Thank you so much for your suggestion.

#2 jerryshelly1

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Posted 26 March 2013 - 06:25 PM

It may not be efficiency of your reaction, although check and make sure you are using compatible buffer, are you sure the activity of your enzyme has not decreased over time (enzyme left at RT O/N).

Consider the molar ratios of your cut insert. If you have a large plasmid, say 6kb, and you are looking for a digested product that is 500bp; the size differences will show concentration differences. Think about how your DNA staining dye works. If you have a large fragment and add EtBr, the EtBr will intercalate in your DNA at more sites than in your small fragment.

#3 phage434

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Posted 26 March 2013 - 07:08 PM

You may be confused by the fact that your insert band is lower in intensity. This is because the intensity is controlled by the mass of the DNA, not by the molarity. The same number of 4000 bp fragments will have a much brighter band than a similar number of 400 bp fragments.

#4 DrLeo

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Posted 26 March 2013 - 07:29 PM

Thanks all.
My insert has 650bp in size.
Thus, there is no problem with my protocol, right?

#5 jerryshelly1

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Posted 26 March 2013 - 07:39 PM

Doesn't appear to be. You can double check your method if you have an additional plasmid that contains your REs with a different product dropout size. Are you using NEB enzymes? What buffer, I just did a quick search and the supplied buffers do not appear to be compatible - I never heard of this new cutsmart buffer though.

#6 DrLeo

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Posted 26 March 2013 - 08:12 PM

I am using Takara REs, and the buffer included in the kits. I think the buffer is compatible for the enzymes because I follow the manual of the company.




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