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 1µg of DNA in 1h at 37°C

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

most enzymes have conc of  10U/µl, but several are also 20U/µl, 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 10-100µl

 

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

Example: 6 clones

                                                                        Mastermix:    6.5x

                        1.5µl plasmid                                              

                        1µl 10xNEBuffer 2                                       6.5µl

                        1µl 10x BSA                                                  6.5µl

                        0.2µl NheI 10U/µl                                        1.3µl

                        0.1µl XhoI 20U/µl                                         0.65µl

                        6.2µl H2O                                                       40.3µl

 

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

 

Anne Schohl, 2008