Team:Washington/GENERAL DIGESTION PROTOCOL

From 2013.igem.org

Revision as of 03:41, 21 September 2013

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2013 UW iGEM team uses Xba1 and Pst1 combinationfor most digestions
XbaI and SpeI together causes 50% inverted insertions and XbaI is less expensive than SpeI.

Digestion:
10-42.5 uL DNA (200ng/uL = 2ug) Use the highest concentration (>50ng/ul) DNA available.*
DI water to 50 uL (33.5ul for single digest or 32.5 uL for double digest)
5uL of 10X buffer (check http://www.neb.com/nebecomm/EnzymeFinderSearchByName.asp for a list of enzymes and associated buffers) Use CutSmart with all ‘HF’ enzymes and XbaI.
0.5 uL BSA (If required. Not required for CutSmart. See above link) **
1uL enzyme 1 (kept on ice or freezer block)
For double digest add:
1uL enzyme 2 (kept on ice)***
Total 50ul
(Enzymes can added last so they go into a 1X buffer concentration)
Mix the reaction *well* by pipetting up and down a few times (to mix the 50% glycerol).
Incubate at 37C for the required amount of time, usually 1 hr. Digests overnight work well.

If double digest is required, check http://www.neb.com/nebecomm/DoubleDigestCalculator.asp for buffer, temperature, BSA specifications.

*1 unit of enzyme is defined as the amount needed to fully digest 1 ug of DNA in a 50 ul reaction in 1 hour. You can calculate how much enzyme and time is needed to do ~10-fold overdigestion.
**CutSmart contains BSA and is otherwise equivalent to Buffer 4 (except for DTT).
***Note that the enzymes are stored in 50% glycerol. The total RXN needs to be no more than 5% glycerol to get effective digestion. Therefore, the volume of enzyme you add should be no more than 10% of the total reaction volume.
****Thawing of buffer goes quickly when partially immersed in water.

See also:
Restriction Digestion - FreeBio


Diagnostic Digest: use 10-20 uL Total