Lidstrom:Digestion with Restriction Enzymes: Difference between revisions
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Back to [[Lidstrom:Protocols|Protocols]] | Back to [[Lidstrom:Protocols|Protocols]] | ||
== Recipe == | == Considerations == | ||
=== background colonies === | |||
* You can finish digesting, ligating, and transformation and discover that all of your colonies are actually uncut plasmid, not your desired construct. If any of the plasmids you digested have the antibiotic you will be selecting for in your transformation, you can have this issue. Ways to reduce this: | |||
** use a plasmid that supports [http://en.wikipedia.org/wiki/Blue_white_screen blue/white screening] | |||
** use a plasmid backbone that has a toxic insert in it | |||
*** If the insert is toxic, cells that are transformed with undigested plasmid won't be viable. These plasmids are propagated in special strains that have special tools to tolerate them | |||
** use gel purification | |||
*** If the digested piece you want is is different enough in size from the undigested plasmid you can purify the digested piece away. This doesn't work 100%, but can help a lot, depending on the efficiency of digestion. | |||
=== How much DNA should I use? === | |||
* It depends on whether you want to gel purify or not. | |||
*** a few thousand ug is good if you are going to gel purify | |||
== Recipe Comments == | |||
* The total volume recommended varies from 20 uL to 50 uL. | |||
* The amount of DNA and reaction volume you should use depends on whether you want to gel purify it or not | |||
* Usually restriction digests are done at 37oC | |||
** The recommended times range from 30 min - 3 hours. If you have the time to let it go longer, do. The only drawbacks of a long digest time are: (1) you are sitting around waiting for it, and the extra time might not even be helping (2) unspecific cutting may occur. | |||
* Heat inactivate the enzymes by heating to 80oC for 20 minutes. | |||
== Sample Recipe == | |||
*Try it quick and imprecise like [http://2011.igem.org/Team:Washington/Protocols/Digestion iGEM 2011 UW] recommends: | *Try it quick and imprecise like [http://2011.igem.org/Team:Washington/Protocols/Digestion iGEM 2011 UW] recommends: | ||
**10 uL DNA ( | **10+ uL DNA ( | ||
*** Want at least 500 ng but more if you want to gel purify. Can elute a miniprep in 38 uL of water and use that whole thing. Don't use miniprep DNA eluted in buffer; only use DNA eluted in water. | |||
**5uL buffer (check http://www.neb.com/nebecomm/EnzymeFinderSearchByName.asp for a list of enzymes and associated buffers) | **5uL buffer (check http://www.neb.com/nebecomm/EnzymeFinderSearchByName.asp for a list of enzymes and associated buffers) | ||
**0.5 uL BSA (if required. See above) | **0.5 uL BSA (if required. See above) | ||
| Line 9: | Line 30: | ||
**water to 50 uL (32.5 uL, add first) | **water to 50 uL (32.5 uL, add first) | ||
**If two enzymes are required, check http://www.neb.com/nebecomm/DoubleDigestCalculator.asp for buffer, temperature, BSA specifications. | **If two enzymes are required, check http://www.neb.com/nebecomm/DoubleDigestCalculator.asp for buffer, temperature, BSA specifications. | ||
Latest revision as of 07:12, 31 March 2013
Back to Protocols
Considerations
background colonies
- You can finish digesting, ligating, and transformation and discover that all of your colonies are actually uncut plasmid, not your desired construct. If any of the plasmids you digested have the antibiotic you will be selecting for in your transformation, you can have this issue. Ways to reduce this:
- use a plasmid that supports blue/white screening
- use a plasmid backbone that has a toxic insert in it
- If the insert is toxic, cells that are transformed with undigested plasmid won't be viable. These plasmids are propagated in special strains that have special tools to tolerate them
- use gel purification
- If the digested piece you want is is different enough in size from the undigested plasmid you can purify the digested piece away. This doesn't work 100%, but can help a lot, depending on the efficiency of digestion.
How much DNA should I use?
- It depends on whether you want to gel purify or not.
- a few thousand ug is good if you are going to gel purify
Recipe Comments
- The total volume recommended varies from 20 uL to 50 uL.
- The amount of DNA and reaction volume you should use depends on whether you want to gel purify it or not
- Usually restriction digests are done at 37oC
- The recommended times range from 30 min - 3 hours. If you have the time to let it go longer, do. The only drawbacks of a long digest time are: (1) you are sitting around waiting for it, and the extra time might not even be helping (2) unspecific cutting may occur.
- Heat inactivate the enzymes by heating to 80oC for 20 minutes.
Sample Recipe
- Try it quick and imprecise like iGEM 2011 UW recommends:
- 10+ uL DNA (
- Want at least 500 ng but more if you want to gel purify. Can elute a miniprep in 38 uL of water and use that whole thing. Don't use miniprep DNA eluted in buffer; only use DNA eluted in water.
- 5uL buffer (check http://www.neb.com/nebecomm/EnzymeFinderSearchByName.asp for a list of enzymes and associated buffers)
- 0.5 uL BSA (if required. See above)
- 1uL enzyme 1
- water to 50 uL (32.5 uL, add first)
- If two enzymes are required, check http://www.neb.com/nebecomm/DoubleDigestCalculator.asp for buffer, temperature, BSA specifications.
- 10+ uL DNA (
