IIT Madras

And then the E.coli said, "Let there be light"

Double Restriction

• Use NEB digest finder to find the buffer that is compatible for the digestion.
• The buffers are 10 X. Thus the volume of the buffer added to the digestion must be such that it is 1 X.
• Usually 0.3-0.5 ul of Enzymes can be used for every 1 ug of DNA – 1 unit of Enzyme is added.
• In case of sequential digestions, the following techniques can be used:
  • Use enzyme 1 – restrict – purify DNA – Use Enzyme 2.
  • Use enzyme which requires a more dilute buffer first, Then add the concentrated buffer for restricting DNA using 2nd Enzyme.
• Analytic Restrictions – final volume 10 ul.
• Preparative Restrictions – 40 – 50 ul.
• BSA should be added if recommended. 0.1 ul of BSA is usually added for 10 ul reactions.
• Run the products on a gel and check double digestion.

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