Set up digests as described above, as if you were going to ligate the plasmid to an insert.
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The sTRAP protocol uses 5% SDS that maximizes protein solubilization. Prepare positive control reaction with template of known cutting site corresponding to the restriction enzyme of choice.
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After the initial.
After trypsin digestion, peptides are analyzed. . A specific protocol for single digestion using this restriction enzyme can be accessed using our free online tool, NEBcloner.
Mix:.
Protocol for DNA Digestion with a Single Restriction Enzyme. 3 µL 10x Buffer. .
. Treat your PCR product ( 50microlitre) with one unit of Dpn1 and incubate at 37C for at least 2 hrs to ensure digestion of all your template DNA.
The standard electroporation protocol was used to transform plasmid into E.
Setting up a Double Digestion with a Unique Buffer (designated “U”) NEB currently supplies three enzymes with unique buffers: EcoRI, SspI and DpnII.
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Select restriction enzymes for your insert and vector, and determine the appropriate reaction buffers. The lysate is neutralized by the addition of acidic potassium acetate ( Buffer P3 ).
Please note that NEBcloner will also provide detailed double digest protocols using this enzyme.
Mix gently and spin down briefly.
. . Briefly centrifuge to settle tube contents.
Mar 24, 2021 · Kits are available for total RNA purification, plasmid miniprep, gel extraction, and DNA & RNA cleanup. Please note that NEBcloner will also provide detailed double digest protocols using this enzyme. . . Incubate at 37 degrees for at least 1 hour.
Proteins are trapped on a borosilicate glass membrane filter, where SDS is subsequently removed from the filter.
. The standard electroporation protocol was used to transform plasmid into E.
Run 5 \(\mu L\) of your digested sample in an agarose gel in order to check that your digestion worked.
By definition: one unit of enzyme will cut 1 µg of DNA in a 50 µL reaction in 1 hour.
We recommend around 100ng of total DNA in a standard ligation reaction.
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