"
Team:Amsterdam/Notebook/Protocols/Digestion
From 2011.igem.org
(→Digestion: Making new constructs) |
|||
| (9 intermediate revisions not shown) | |||
| Line 2: | Line 2: | ||
=Digestion: Making new constructs= | =Digestion: Making new constructs= | ||
| - | We used this protocol for digesting | + | We used this protocol for digesting the biobricks. |
| - | + | <br><br> | |
==Materials== | ==Materials== | ||
*PCR tubes | *PCR tubes | ||
| Line 10: | Line 10: | ||
*BSA | *BSA | ||
*Enzyme Buffer (NEBuffer 2) | *Enzyme Buffer (NEBuffer 2) | ||
| - | |||
| - | < | + | <i>Note: All materials should be kept on ice during preparation.</i> |
| + | <br><br> | ||
==Protocol== | ==Protocol== | ||
| Line 70: | Line 70: | ||
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> | ||
==Double digest== | ==Double digest== | ||
| Line 84: | Line 85: | ||
7. take 1 μl of each undigested sample.<br> | 7. take 1 μl of each undigested sample.<br> | ||
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> | ||
| - | + | <br><br> | |
=Digestion: Changing the backbone= | =Digestion: Changing the backbone= | ||
We used this protocol to transfer the construct in a new backbone. | We used this protocol to transfer the construct in a new backbone. | ||
| - | + | <br><br> | |
==Materials== | ==Materials== | ||
*PCR tubes | *PCR tubes | ||
| Line 94: | Line 95: | ||
*BSA | *BSA | ||
*Enzyme Buffer (NEBuffer 2) | *Enzyme Buffer (NEBuffer 2) | ||
| - | < | + | <i>Note: All materials should be held on ice during preparation.</i> |
| - | + | <br><br> | |
| - | Note: All materials should be held on ice during preparation. | + | |
| - | + | ||
==Protocol== | ==Protocol== | ||
{| border="1" | {| border="1" | ||
| - | !align="left"| | + | !align="left"|Sample |
| - | !align="right"| | + | !align="right"|μl |
|- | |- | ||
|DNA | |DNA | ||
| Line 123: | Line 122: | ||
|- style="font-style:italic;" | |- style="font-style:italic;" | ||
|Total | |Total | ||
| - | |align="right"|20 | + | |align="right"|20 μl |
|} | |} | ||
<br/> | <br/> | ||
| - | 1. Incubate the restriction digest at | + | 1. Incubate the restriction digest at 37°C for 2 hours.<br> |
| - | 2. Incubate at | + | 2. Incubate at 80°C for 20 min to heat kill the enzymes.<br> |
| - | 3. Store at - | + | 3. Store at -4°C. |
| - | + | ||
| + | <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> | ||
1. First use only EcoRI.<br> | 1. First use only EcoRI.<br> | ||
| - | 2. Incubate at 37 | + | 2. Incubate at 37°C for 1 hour.<br> |
| - | 3. take 1 | + | 3. take 1 μl of each sample.<br> |
4. add PstI to all the samples.<br> | 4. add PstI to all the samples.<br> | ||
| - | 5. Incubate at 37 | + | 5. Incubate at 37°C for 1 hour.<br> |
| - | 6. take 1 | + | 6. take 1 μl of each sample.<br> |
| - | 7. take 1 | + | 7. take 1 μl of each undigested sample.<br> |
| - | 8. fill each of the three samples up to 10 | + | 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.
