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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

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

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.

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