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Richard Lab:Restriction Digest: Difference between revisions
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Richard Lab:Restriction Digest (view source)
Revision as of 13:26, 18 January 2011
, 18 January 2011→Procedure
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==Procedure== | ==Procedure== | ||
1. Quickly vortex all ingredients (Buffer, BSA, DNA) before beginning. | |||
2. Add the following in a micro-centrifuge tube: | |||
::*5μl of Buffer (usually NEBuffer 2); | |||
::*1μl of BSA; | ::*1μl of BSA; | ||
::*0.5 picomoles DNA (see [[DNA Quantification]]);[[user:Michael A. Speer | Mike]] normally uses 10μL of miniprep or 5μL of purified PCR product. | |||
::*Water to make 48μl. | ::*Water to make 48μl. | ||
3. Vortex Enzymes and add 1μl of each to the tube. | |||
::*If you're digesting purified PCR product (i.e. "insert"), add 1μl of DpnI to the reaction. | ::*If you're digesting purified PCR product (i.e. "insert"), add 1μl of DpnI to the reaction. | ||
4. Incubate reaction in a 37°C water bath for at least one hour. | |||
::*If your digesting a "vector", add 1μl Antarctic Phosphatase and 6μl of Phosphatase buffer after 2 hours of incubation and incubate for another hour. | ::*If your digesting a "vector", add 1μl Antarctic Phosphatase and 6μl of Phosphatase buffer after 2 hours of incubation and incubate for another hour. | ||
::*See the [[Richard Lab:Bio-Brick Assembly Schedule|Bio-Brick Assembly Schedule]] for more details. | ::*See the [[Richard Lab:Bio-Brick Assembly Schedule|Bio-Brick Assembly Schedule]] for more details. | ||
5. Heat kill the digests for 20 minutes at 80°C. | |||
6. Store digested DNA in the refrigerator (4°C)for use in the very near future. | |||
==Notes== | ==Notes== | ||
