From 2011.igem.org
(Difference between revisions)
|
|
| (2 intermediate revisions not shown) |
| Line 1: |
Line 1: |
| - | {{:Team:DTU-Denmark/Templates/Standard_page_begin|Restriction/Digestion}}
| |
| | | | |
| - | == Restriction/Digestion ==
| |
| - | Materials:
| |
| - | * 25 µL DNA (but varies depending on your DNA conc.)
| |
| - | * 10 µL FD-buffer
| |
| - | * 1 µL restriction enzyme (1 & 2) (you can also use 2.5µL of each RE, which showed good results in previous digestions (AJ+PF))
| |
| - | * Using 1µL RE: 63ul ddH2O. Using 2.5µL RE: 60 µL ddH2O (Total volume of 100 µL)
| |
| - |
| |
| - | # Turn the thermo block on 30 min before use. Mix everything – add the enzymes last. Leave for 2 hour at 37 C in thermo block/incubator.
| |
| - | # To deactivate the enzyme leave it at 65 C in a thermo block for 10 min.
| |
| - | # Keep on ice until its cooled down.
| |
| - | IMPORTANT: If you only use one RE on your plasmid
| |
| - | SAP treatment is required:
| |
| - | * Mix 50µL of the restricted DNA with 2.5µL SAP and 5µL SAP-buffer
| |
| - | * Put it in the incubator 37°C for 60 min
| |
| - | * 15 minutes on heat block 65°C to deactivate enzyme
| |
| - | * Place on ice
| |
| - |
| |
| - | Enzyme selection for BioBricks digest
| |
| - | A BioBricks part:
| |
| - |
| |
| - | -E--X---Part---S--P-
| |
| - |
| |
| - | Vector with upstream part - S,P
| |
| - | Downstream Insert - X,P
| |
| - | Upstream Insert - E,S
| |
| - | Vector with downstream part - E,X
| |
| - |
| |
| - | E= EcoRI X= XbaI S= SpeI P= PstI
| |
| - |
| |
| - | Enzymes should be kept in the icebox from the freezer at all times.
| |
| - |
| |
| - |
| |
| - |
| |
| - | {{:Team:DTU-Denmark/Templates/Standard_page_end}}
| |
Latest revision as of 19:19, 21 September 2011
"
