Restriction Enzyme Buffer D, 10X, is specially designed for use in double digestions.
Quality Control:
Functional Test:
Functionaly tested in DNA digestion with 15 randomly picked conventional restriction endonucleases. Digestion conditions are per activity assay of enzymes. Reaction products analyzed by agarose electrophoresis and ethidium bromide staining. No change in digestion pattern is observed compared to previous lot.
Endo- and Exonucleases Assay:
Single stranded and double standed radiolabeled oligonucleotides are incubated with the 1X buffer at 37 degrees C for 16 hours, separated on polyacrylamide gel and analyzed by phosphoimaging. No signs of radiolabeled oligo degradation is considered as the absence of endo- and exonucleases activities.
Nicking Activities Assay:
Supercoiled pUC19 DNA is incubated with the 1X buffer at 37 degrees C for 17 hours and analyzed by agarose gel electrophoresis and ethidium bromide staining. There is no linearised plasmid form and relaxation of supercoiled plasmid form compared to negative control.
Storage and Stability:
May be stored at 4 degrees C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20 degrees C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.
Note: This buffer contains BSA. To avoid the risk of bacterial growth, do not store buffers containing BSA at 4 degrees C for more than one week.
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