Latest revision as of 11:09, 2 October 2011

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Double Restriction

Common Protocol

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.

@@ Line 76: / Line 76: @@
 <li>The buffers are 10 X. Thus the volume of the buffer added to the digestion must be such that it is 1 X.</li>
 <li>Usually 0.3-0.5 ul of Enzymes can be used for every 1 ug of DNA – 1 unit of Enzyme is added.</li>
-<li>4. In case of sequential digestions, the following techniques can be used:
+<li>In case of sequential digestions, the following techniques can be used:
 <ul><li>Use enzyme 1 – restrict – purify DNA – Use Enzyme 2.</li>
-<li>Use enzyme  which requires a more dilute buffer first, Then add the concentrated buffer for restricting DNA using 2nd Enzyme.</li>
+<li>Use enzyme  which requires a more dilute buffer first, Then add the concentrated buffer for restricting DNA using 2nd Enzyme.</li></ul>
 </li>
 <li>Analytic Restrictions – final volume 10 ul.</li>
 <li>Preparative Restrictions – 40 – 50 ul.</li>
 <li>BSA should be added if recommended. 0.1 ul of BSA is usually added for 10 ul reactions.</li>
+<li>Run the products on a gel and check double digestion.</li>
 </ul>
 </div>