Digestion

From 2008.igem.org

Revision as of 00:58, 30 October 2008 by Lphalen (Talk | contribs)
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

PrincetonLogo.gif

PRINCETON IGEM 2008

Home Project Overview Project Details Experiments Results Notebook
Parts Submitted to the Registry Modeling The Team Gallery




Digestion Protocol

A Digestion is used to cut a plasmid at a specific site.


1. Use X units of enzymes per 1ug of DNA (over digesting by factor of X)


Usually we use over digest factor of 10 unless otherwise specified in the enzyme tech sheet. If over digestion results in star activity use 3X.


Total volume must be greater than or equal to 10X volume of enzyme.


3. Calculate the volume required for each

DNA µg = DNA volume * concentration

Enzyme volume = Enzyme unit/µl* # units = X [µl]

Buffer is dilution factor x dilution of the total volume.

[i.e. for 10X over digest - buffer is 10%, 3x - 30% of total volume]


6. Order of filling

• DNA

• Water

• Buffer

• Enzyme


7. Incubate for 3 hours at the specified temperature for the enzyme and deactivate at the appropriate temperature for 20 mins.


8. Please keep the buffer on ice at all times when out of -20C. Keep the enzyme in the benchtop coolers at all times when out of the -20C.