Cfrench:bbcloning

5. Cloning a PCR product into a Biobrick vector
This section assumes that your PCR product is a Biobrick or Edinbrick. If not, you will have to adjust the procedure according to the restriction sites on your primers.

First, choose a pair of enzymes, and select the appropriate buffer. The buffers listed here are from Promega, but any other supplier’s buffers should do equally well; if using non-Promega buffers, consult the technical appendix of the manufacturer’s catalog to determine which buffers can be used for which enzyme combinations.

Typical enzyme combinations for cloning biobricks:

 * SacI/SpeI (Edinbricks only): buffer E or multi-core
 * XbaI/PstI: buffer H
 * EcoRI/SpeI: buffer E
 * EcoRI/PstI: buffer H


 * Next, choose a vector. If using SacI/SpeI you must use an Edinbrick vector. For general purposes, use Edinbrick1, which contains a lacZ’ marker gene allowing blue-white selection. If your PCR product includes lacZ’, then you can use Edinbrick2 (xylE marker gene) or Edinbrick3 (idoA marker gene).

If you are using an enzyme combination other than SacI/SpeI, you can use a non-Edinbrick Biobrick vector such as J33202; however, Edinbrick vectors will also work and are recommended for routine purposes unless you need a resistance marker other than ampicillin.


 * Set up a restriction digest in a 1.5 ml microcentrifuge tube as follows:

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 * water: 8 microlitres
 * vector DNA: 4 microlitres
 * PCR product: 4 microlitres
 * 10 x buffer: 2 microlitres
 * enzyme A: 1 microlitre
 * enzyme B: 1 microlitre
 * total volume: 20 microlitres.


 * Mix gently, spin briefly in a microcentrifuge to make sure that all the liquid is at the bottom of the tube, and incubate at 37˚C for 2 hours or more.


 * Purify the DNA from the digest using the glass bead procedure in section 4, with the following modifications: use 65 microlitres of 6 M sodium iodide, 5 microlitres of glass beads, and elute into 10 microlitres of EB.


 * To the 10 microlitres of eluted DNA, add 1.2 microlitres of 10 x DNA ligase buffer and 1.2 microlitres of T4 DNA ligase. Mix gently and spin briefly. Incubate at 16˚C overnight. There is as 16˚C waterbath in the working cold room.


 * Next, transform competent E. coli cells (JM109, DH5α or a similar strain) as described in section 6. Use 5 microlitres of the ligation.


 * This procedure will generate two main colony types – those with the unaltered vector, and those in which the marker gene has been replaced by the new insert. You can distinguish between these colony types as follows:


 * If using Edinbrick 1 as a vector, plate the cells on Blue-White selection plates (containing 90 mg/l IPTG and 40 mg/l Xgal). Blue colonies will be Edinbrick1 vector, white colonies may be the desired construct.


 * If using Edinbrick 2, plate on plain ampicillin plates. After colonies have grown, you can then test individual colonies by adding a drop of (reasonably fresh) 10 mM catechol to each colony. Colonies which go yellow after a few minutes are vector colonies. Colonies which do not go yellow may be the desired construct. This does not always work well with small colonies, so it may be necessary to patch the cells and test the patches.


 * If using Edinbrick 3, plate on plain ampicillin plates. Colonies with the unaltered Edinbrick 3 vector should go brownish blue due to oxidation of indole. This reaction may be quite slow, but may be speeded up by adding a little indole to the plate. This does not always work well with primary colonies, but patches give good results after overnight incubation. As I write this, Edinbrick 3 has only just been developed, so we are still experimenting with it. If we develop a good protocol, Edinbrick 3 may supercede Edinbricks 1 and 2 for routine purposes.

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