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Team:INSA Toulouse/contenu/lab practice/notebook/protocols/digest
From 2013.igem.org
Notebook
Protocols
Digest Protocols
- Our protocol
| DNA | Enzymes | RNAse | 10X Buffer | H2O |
| 5µL | 0.5µL of each (1µL in total) | 0.1µL | 1µL (10% of total volume) | 2µL (adjust at 10µL) |
Do not overreach 10 % Volume with enzymes.
- iGEM Restriction Digest Protocol
estimated time: 30 min. active, 50 min. incubation
The following protocol assumes you'll be doing restriction digests for 3A assembly, therefore we refer to your digests as:
· Part A (The 1st part in the future composite part)
· Part B (The 2nd part in the future composite part)
· Linearized plasmid backbone (The destination plasmid backbone for your composite part)
If you are simply doing a restriction digest for quality control, you can use the protocol below.
Restriction Digest from iGEM Videos.
Materials
· Ice and bucket/container
· (1) 8-tube strip, or (3) 0.6ml thin-walled tubes
· Part A (Purified DNA, > 16ng/µl)
· Part B (Purified DNA, > 16ng/µl)
· Linearized plasmid backbone (25ng/µl)
· dH2O
· NEB Buffer 2
· BSA
· Restriction Enzymes: EcoRI, SpeI, XbaI, PstI, DpnI
· Thermal cycler
Notes
· You should keep all materials on ice.
· At iGEM HQ we use restriction enzymes from New England Biolabs
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