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1. Where can I obtain BioBrick parts?

If you are at an institute that takes part in the iGEM competition, your institute may have a copy of the BioBrick Parts Registry. If not, you can make them yourself via PCR or de novo DNA synthesis. Be sure and check out Ginkgo's part design tool that will automatically generate primers for construction of BioBrick parts by PCR.

No. It is only necessary for the destination plasmid to have a different antibiotic resistance than the plasmids containing the parts you are assembling. Even this constraint is only necessary if you want to avoid doing a gel purification on the digested input parts.

First you should check if your competent cells are actually competent by doing a control transformation with some purified pUC19 plasmid DNA (you should get at least 1E7 transformants per μg of plasmid DNA). Next, make sure that the antibiotic in your plates matches the antibiotic of your destination plasmid. If you have performed the suggested checks on your transformation and you still aren't getting any colonies, it is likely that there is a problem with your Assembly. If that is the case you should next confirm that the digest reaction was successful by running some of your undigested and digested DNA fragments on an agarose gel to make sure the digest is working and that the products of the digest are the correct length. See Questions 5 and 6 below on debugging a digest reaction. If you have already confirmed that your digest is working correctly, you should check that your ligation is working correctly. See Question 7 below for further information on debugging a ligation reaction.

It appears that your restriction digest isn't working and you should begin by checking that you have the DNA you think you have by sequencing some of your purified, undigested DNA. If you are sure you have the correct DNA, you should check that your reagents are working and that you are using the correct reaction conditions. NEB provides useful support information for their restriction enzymes, BSA, and NEBuffer 2.

You should begin by checking that you have the DNA you think you have by sequencing some of your purified, undigested DNA. If you are sure you have the correct DNA, one or both of your restriction enzymes may not be cutting. Consider doing two single digests with the two enzymes you want to use to see which, if either of them, isn't cutting.

You should check that your ligation reaction is working. NEB provides useful support information including debugging experiments on the respective product pages for T4 DNA ligase and T4 DNA ligase reaction buffer. Common problems include failing to resuspend the precipitate in the T4 DNA ligase reaction buffer or using old buffer where the ATP has degraded due to repeated freeze/thaw cycles or where the DTT has broken down (the buffer should smell strongly if the DTT is still ok). Note that in a 50μl reaction you should not add more than 10ng of any of the DNA fragments, a more concentrated ligation doesn't always produce more correct ligation products.

NEB sells the BioBrick Assembly Kit which contains all the necessary reagents.

If you follow the instructions in the BioBrick Assembly Manual, any waste can be disposed of in regular trash. Note that this is not the case for any cells used during the transformation stage, these must be disposed of in biohazard waste and autoclaved in a manner required by any relevant local regulations. Also, any pipet tips used should be disposed of in sharps waste in a manner required by any relevant local regulations.

Still looking for an answer to your question?

For questions relating to BioBrick assembly, please contact Ginkgo BioWorks Customer Support.

For questions relating to the enzymes and buffers in the BioBrick Assembly Kit, please contact NEB at

• Email: info@neb.com
• Tel.: 1-800-632-5227