Why isn't SacI cutting?

1. The most frequent problem encountered is salt inhibition. DNA preparations can have high salt concentrations. If the volume of DNA added to the reaction is a high percentage of the total volume, salt can inhibit cutting. Increasing the reaction volume may help. The DNA may need to be ethanol precipitated and washed with 70% ethanol to reduce the salt. Drop dialysis is the most effective way of reducing the salt.
2. High pH will inhibit SacI.
3. Supercoiled plasmids may require up to 5-fold more SacI for complere digestion than linear DNA.
4. SacI is inhibited by common clinical anticoagulants found in some preparation of anticoagulated peripheral blood and bone marrow. Levels of EDTA and ACD (citric acid-sodium citrate-dextrose) in standard sample preparation have been shown to inhibit SacI. Three times the normal concentration for hearin is required to inhibit SacI. (Rebase ref# 2886 Coad, J.e., Lander, T.A., Litz, C.E. "Inhibition of restriction endonucleases by common clinical anticoagulants" Anal. Biochem. 205: 368-369 (1992).)

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