Restriciton Digest

 

Basic rules:

Amount of enzyme should never exceed 1/10 of reaction volume, might result in star activity

 

1 Unit of enzyme cuts 1g of DNA in 1h at 37C

use 1.5 - 2x more enzyme than necessary, e.g. for 5g of DNA use 7.5-10U of enzyme

most enzymes have conc of  10U/l, but several are also 20U/l, check before using them, adding too much enzyme might result in star activity as well even if volume is lower than 1/10

 

use reaction volume between 10-100l

 

some enzymes need BSA in reaction, prepare 10x BSA from 100x BSA stock, use like NEB Restriction enzyme buffers

 

 

 

Restriction digest for screening Mini-prep DNA:

 

Use 1-2l of Mini-Prep DNA and 0.1-0.2l of Restriction enzyme

Example: 6 clones

                                                                        Mastermix:    6.5x

                        1.5l plasmid                                              

                        1l 10xNEBuffer 2                                       6.5l

                        1l 10x BSA                                                  6.5l

                        0.2l NheI 10U/l                                        1.3l

                        0.1l XhoI 20U/l                                         0.65l

                        6.2l H2O                                                       40.3l

 

add 8.5l Mastermix to 1.5l plasmid for 10l final volume, incubate 1h at 37C, load everything on agarose gel

 

Anne Schohl, 2008