"
Team:Washington/GENERAL DIGESTION PROTOCOL
From 2013.igem.org
(Difference between revisions)
(→') |
|||
| Line 1: | Line 1: | ||
{{:Team:Washington/WA_Template}} | {{:Team:Washington/WA_Template}} | ||
| - | |||
| - | |||
| - | |||
| - | |||
| Line 11: | Line 7: | ||
<b>General Digestion Protocol in PCR Tubes</b> | <b>General Digestion Protocol in PCR Tubes</b> | ||
</header> | </header> | ||
| - | |||
<body> | <body> | ||
| - | |||
2013 UW iGEM team uses Xba1 and Pst1 <a href = "https://www.neb.com/tools-and-resources/interactive-tools/double-digest-finder?enzyme1=%7B2DAF5F0E-59C4-4A5D-8809-6CEAD6BAE4B4%7D&enzyme2=%7B90EFE184-8442-45F0-8286-04CA22EEFFF2%7D">combination</a>for most digestions | 2013 UW iGEM team uses Xba1 and Pst1 <a href = "https://www.neb.com/tools-and-resources/interactive-tools/double-digest-finder?enzyme1=%7B2DAF5F0E-59C4-4A5D-8809-6CEAD6BAE4B4%7D&enzyme2=%7B90EFE184-8442-45F0-8286-04CA22EEFFF2%7D">combination</a>for most digestions | ||
Latest revision as of 04:32, 27 September 2013
General Digestion Protocol in PCR Tubes 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
15 ul 25 ul 10-20 uL of DNA
2.5uL 10x Buffer
(0.5uL of BSA)
0.5 - 10 uL of Enz 1
0.5 - 10 uL of Enz 2
Fill to 25 uL with H20
25 uL Total11 uL of DNA
1.5 uL of buffer 10x
(0.5 uL of BSA)
0.5 - 1.0 uL of Enz 1
0.5 - 1.0 uL of Enz 2
15 uL Total
