Team:British Columbia/Protocols/Restriction Digests
From 2012.igem.org
(Difference between revisions)
(Created page with "{{Template:Team:British Columbia Header}}") |
|||
Line 1: | Line 1: | ||
{{Template:Team:British Columbia Header}} | {{Template:Team:British Columbia Header}} | ||
+ | |||
+ | =Restriction Digest Protocol= | ||
+ | |||
+ | 1. Prepare reaction mix, following the basic ratios below. Each reaction requires at least 4 uL of master mix. | ||
+ | |||
+ | Notes: | ||
+ | *do not add DPN1 when working with plasmid DNA - it will cut all methylated DNA. | ||
+ | *keep the restriction enzymes as close to -20°C as possible. | ||
+ | 2. Add 4 uL of master mix and 4 uL of undigested template DNA (plasmid or PCR product) to a 200 uL PCR tube. | ||
+ | |||
+ | 3. Heat the mixtures at 37°C for 30 minutes for optimal enzyme activity, then 80°C for 20 minutes to inactivate the enzymes. | ||
+ | *easiest to use a thermocycler |
Revision as of 00:33, 26 June 2012
![UBC iGEM 2012 header](https://static.igem.org/mediawiki/2012/f/f5/IGEMUBC2012Header.jpg)
Restriction Digest Protocol
1. Prepare reaction mix, following the basic ratios below. Each reaction requires at least 4 uL of master mix.
Notes:
- do not add DPN1 when working with plasmid DNA - it will cut all methylated DNA.
- keep the restriction enzymes as close to -20°C as possible.
2. Add 4 uL of master mix and 4 uL of undigested template DNA (plasmid or PCR product) to a 200 uL PCR tube.
3. Heat the mixtures at 37°C for 30 minutes for optimal enzyme activity, then 80°C for 20 minutes to inactivate the enzymes.
- easiest to use a thermocycler