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Team:Amsterdam/Notebook/Protocols/Digestion
From 2011.igem.org
Contents |
Digestion: Making new constructs
We used this protocol for the digestion of the biobricks.
Materials
- PCR tubes
- dH20
- Enzymes (EcoRI, XbaI, SpeI, PstI)
- BSA
- Enzyme Buffer (NEBuffer 2)
Note: All materials should be held on ice during preparation.
Protocol
| Vector | ul |
|---|---|
| DNA | 10 |
| NEB buffer 2 | 2 |
| BSA | 0,5 |
| SpeI | 1 |
| PstI | 1 |
| H2O | 5,5 |
| Total | 20 ul |
| Insert | ul |
|---|---|
| DNA | 10 |
| NEB buffer 2 | 2 |
| BSA | 0,5 |
| XbaI | 0,5 |
| PstI | 1 |
| H2O | 6 |
| Total | 20 ul |
1. Incubate the restriction digest at 37C for 2 hours.
2. Incubate at 80C for 20min to heat kill the enzymes.
3. Store at -4C.
Double digest
To check if the enzymes digest the plasmid as expected, the following digestion should be performed:
1. First use only SpeI(vector) and XbaI(insert).
2. Incubate at 37 degrees Celsius for 1 hour.
3. take 1 ul of each sample.
4. add PstI to all the samples.
5. Incubate at 37 degrees Celsius for 1 hour.
6. take 1 ul of each sample.
7. take 1 ul of each undigested sample.
8. fill each of the three samples up to 10 ul including LD and check them on agarose gel.
Digestion: Changing the backbone
We used this protocol to transfer the construct in a new backbone.
Materials
- PCR tubes
- dH20
- Enzymes (EcoRI, PstI)
- BSA
- Enzyme Buffer (NEBuffer 2)
Note: All materials should be held on ice during preparation.
Protocol
| sample | ul |
|---|---|
| DNA | 10 |
| NEB buffer 2 | 2 |
| BSA | 0,5 |
| EcoRI | 0,5 |
| PstI | 1 |
| H2O | 6 |
| Total | 20 ul |
1. Incubate the restriction digest at 37C for 2 hours.
2. Incubate at 80C for 20min to heat kill the enzymes.
3. Store at -4C.
Double digest
To check if the enzymes digest the plasmid as expected, the following digestion should be performed:
1. First use only EcoRI.
2. Incubate at 37 degrees Celsius for 1 hour.
3. take 1 ul of each sample.
4. add PstI to all the samples.
5. Incubate at 37 degrees Celsius for 1 hour.
6. take 1 ul of each sample.
7. take 1 ul of each undigested sample.
8. fill each of the three samples up to 10 ul including LD and check them on agarose gel.
