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Restriction Enzyme Digests
From 2010.igem.org
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http://openwetware.org/wiki/Endy:Restriction_Digest | http://openwetware.org/wiki/Endy:Restriction_Digest | ||
| - | + | ==Materials== | |
| + | |||
| + | *Restriction enzymes ([http://www.neb.com/nebecomm/products/productR0101.asp EcoR I], [http://www.neb.com/nebecomm/products/productR0133.asp Spe I], [http://www.neb.com/nebecomm/products/productR0145.asp Xba I] or [http://www.neb.com/nebecomm/products/productR0140.asp Pst I]) from [http://www.neb.com/ NEB] | ||
| + | *NEB2 buffer | ||
| + | *BSA | ||
| + | *Nuclease free H<sub>2</sub>O | ||
| + | ==Digest Mix== | ||
| + | |||
| + | ''Example - 50 μL reaction. 100 μL reactions are also common especially if your DNA to be cut is dilute.'' | ||
| + | |||
| + | *5 μL NEB2 buffer | ||
| + | *X μL DNA ( ~500 ng). | ||
| + | *0.5 μL 100X BSA | ||
| + | *1 μL BioBricks enzyme 1 | ||
| + | *1 μL BioBricks enzyme 2 | ||
| + | *(42.5 - X) μL deionized, sterile H<sub>2</sub>O | ||
| + | |||
| + | |||
| + | ==Digestion== | ||
| + | # Digest at 37 degrees for 1.5 hours | ||
| + | # Heat kill for 20 minutes | ||
| + | # Add 1 μL Phosphotase to backbone | ||
| + | # Incubate for 30 minutes @ 37 degrees | ||
| + | # heat kill @80 degrees for 20 minutes. | ||
| + | |||
| + | Back to [[Team:Northwestern/Protocol|<span style="color:#4B088A;">Protocol</span>]] | ||
| + | [[Category:Protocol]] | ||
Latest revision as of 21:05, 16 October 2010
Contents |
CHOOOSE
http://openwetware.org/wiki/Knight:Restriction_Digest
http://openwetware.org/wiki/Endy:Restriction_Digest
Materials
Digest Mix
Example - 50 μL reaction. 100 μL reactions are also common especially if your DNA to be cut is dilute.
- 5 μL NEB2 buffer
- X μL DNA ( ~500 ng).
- 0.5 μL 100X BSA
- 1 μL BioBricks enzyme 1
- 1 μL BioBricks enzyme 2
- (42.5 - X) μL deionized, sterile H2O
Digestion
- Digest at 37 degrees for 1.5 hours
- Heat kill for 20 minutes
- Add 1 μL Phosphotase to backbone
- Incubate for 30 minutes @ 37 degrees
- heat kill @80 degrees for 20 minutes.
Back to Protocol
