User:Burak Yilmaz/Notebook/YR10 protein interaction/2009/09/02: Difference between revisions
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===restricton digest for plasmid detection=== | ===restricton digest for plasmid detection=== | ||
==Restriction Digest, using New England Biolabs (NEB) Enzymes== | |||
* Pick a volume where it will be easy to calculate the quantities of 10x and 100x buffers to add and write out a quick recipe (See samples). | * Pick a volume where it will be easy to calculate the quantities of 10x and 100x buffers to add and write out a quick recipe (See samples). | ||
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* Run a bit of the digest sample against the undigested control to confirm that the digstion worked. | * Run a bit of the digest sample against the undigested control to confirm that the digstion worked. | ||
==Sample single digest (50 ul total volume)== | |||
1. 30ul DNA | 1. 30ul DNA | ||
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3. 5ul 10X Buffer | 3. 5ul 10X Buffer | ||
4. 0.5ul 100X BSA | 4. 0.5ul 100X BSA | ||
5. 1ul Restriction Enzyme | 5. 1ul Restriction Enzyme | ||
==Sample double digest (50 ul total volume)== | ==Sample double digest (50 ul total volume)== |
Revision as of 12:11, 3 September 2009
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restricton digest for plasmid detectionRestriction Digest, using New England Biolabs (NEB) Enzymes
(You can add a little water or EB buffer to make this easy, without hurting the reaction)
(Keep a few micoliters to run on a gel against the product, as a negative control)
(See the enzyme(s) page in the NEB manual or website for the correct one)
==Sample single digest (50 ul total volume)== 1. 30ul DNA 2. 13.5ul Water 3. 5ul 10X Buffer 4. 0.5ul 100X BSA 5. 1ul Restriction Enzyme Sample double digest (50 ul total volume)First, check the NEB manual double digest page for optimal conditions or whether it is recommended against for your enzymes 1. 30ul DNA 2. 12.5ul Water 3. 5ul 10X Buffer (Check NEB double digest table - many not be what is used for the single digests 4. 0.5ul 100X BSA (Add if it is required for either enzyme) 5. 1ul Restriction Enzyme 1 6. 1ul Restriction Enzyme 2 reference:http://openwetware.org/wiki/IGEM:Harvard/2009/General_Protocols |