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Team:Amsterdam/Notebook/Protocols/Digestion
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1. Incubate the restriction digest at 37°C for 2 hours.<br> | 1. Incubate the restriction digest at 37°C for 2 hours.<br> | ||
| - | 2. Incubate at 80°C for 20 min to | + | 2. Incubate at 80°C for 20 min to inactivate T4 ligase.<br> |
3. Store at -4°C. | 3. Store at -4°C. | ||
<br><br> | <br><br> | ||
| + | |||
==Double digest== | ==Double digest== | ||
To check if the enzymes digest the plasmid as expected, the following digestion should be performed:<br> | To check if the enzymes digest the plasmid as expected, the following digestion should be performed:<br> | ||
Revision as of 14:12, 19 September 2011
Contents |
Digestion: Making new constructs
We used this protocol for digesting the biobricks.
Materials
- PCR tubes
- dH20
- Enzymes (EcoRI, XbaI, SpeI, PstI)
- BSA
- Enzyme Buffer (NEBuffer 2)
Note: All materials should be kept on ice during preparation.
Protocol
| Vector | μl |
|---|---|
| DNA | 10 |
| NEB buffer 2 | 2 |
| BSA | 0,5 |
| SpeI | 1 |
| PstI | 1 |
| H2O | 5,5 |
| Total | 20 μl |
| Insert | μl |
|---|---|
| DNA | 10 |
| NEB buffer 2 | 2 |
| BSA | 0,5 |
| XbaI | 0,5 |
| PstI | 1 |
| H2O | 6 |
| Total | 20 μl |
1. Incubate the restriction digest at 37°C for 2 hours.
2. Incubate at 80°C for 20 min to inactivate T4 ligase.
3. Store at -4°C.
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°C for 1 hour.
3. take 1 μl of each sample.
4. add PstI to all the samples.
5. Incubate at 37°C for 1 hour.
6. take 1 μl of each sample.
7. take 1 μl of each undigested sample.
8. fill each of the three samples up to 10 μl 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 | μl |
|---|---|
| DNA | 10 |
| NEB buffer 2 | 2 |
| BSA | 0,5 |
| EcoRI | 0,5 |
| PstI | 1 |
| H2O | 6 |
| Total | 20 μl |
1. Incubate the restriction digest at 37°C for 2 hours.
2. Incubate at 80°C for 20 min to heat kill the enzymes.
3. Store at -4°C.
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°C for 1 hour.
3. take 1 μl of each sample.
4. add PstI to all the samples.
5. Incubate at 37°C for 1 hour.
6. take 1 μl of each sample.
7. take 1 μl of each undigested sample.
8. fill each of the three samples up to 10 μl including LD and check them on agarose gel.
Purification
The samples should be purified after digestion. We have done this with a gel purification kit.
Both the gel extraction protocols/kits from [http://www.qiagen.com/hb/qiaquickgelextractionkit_en Qiagen] and [http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_k0691.pdf Fermentas] were used.
