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Team:Amsterdam/Notebook/Protocols/Digestion
From 2011.igem.org
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*Enzyme Buffer (NEBuffer 2) | *Enzyme Buffer (NEBuffer 2) | ||
| - | <i>Note: All materials should be | + | <i>Note: All materials should be kept on ice during preparation.</i> |
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==Protocol== | ==Protocol== | ||
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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 enzymes.<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> | ||
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8. fill each of the three samples up to 10 μl including LD and check them on agarose gel.<br> | 8. fill each of the three samples up to 10 μl including LD and check them on agarose gel.<br> | ||
| - | + | =Purification= | |
| - | + | Samples are often purified after digestion using a gel extraction kit. Gel extraction protocols and 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.<br><br> | |
{{:Team:Amsterdam/Footer}} | {{:Team:Amsterdam/Footer}} | ||
Latest revision as of 23:59, 21 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 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 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
Samples are often purified after digestion using a gel extraction kit. Gel extraction protocols and 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.
