Design questions:

Question: How can I use PCR to add overlapping regions to my gBlocks™ Gene Fragments that I will use in a Gibson Assembly reaction?

Answer: Don’t use PCR to add the overhangs, because PCR can preferentially amplify smaller byproducts in the prep, leading to issues with the cloning reaction and increasing the number of colonies you need to screen to find the correct full-length insert.

Instead, build the overhangs into the gBlocks fragment itself. The amount of gBlocks fragments you will receive is more than enough for a couple of assembly reactions, so the additional PCR step is not required to generate additional material and just adds an additional enzymatic step to the protocol that can introduce errors.

Question: Can I do a restriction digest directly on my gBlocks Gene Fragment prep?

Answer: Yes, the yield provided is more than sufficient for a standard digestion/ligation reaction. You will need to add the restriction sites to each end of your gBlocks Gene Fragment plus 6 additional flanking bases. Many restriction sites need a short DNA stretch upstream of the recognition site to grab onto, to digest efficiently. You can use any 6 bp sequence that has balanced GC content and is not repetitive.

Question: What should I do if the IDT online ordering tool says that my sequence is too complex to order, but I need it as is?

Answer: In most cases, our system has found a region with either very high G/C content or several small repeats that would prevent us from synthesizing your sequence.

• If your sequence contains a coding region, you can use codon optimization to reduce the complexity. Codon optimization uses synonymous codons to retain the final amino acid sequence that is expressed while generating a sequence that we can synthesize.
• If your sequence is not a coding region, we might be able to synthesize the sequence as a custom gene, which is delivered in a basic pUC vector, rather than just a linear, dsDNA gBlocks fragment. For assistance, send your sequences and any questions to [email protected].

Usage questions:

Question: What can I do if I do not get any colonies during my cloning reaction?

Answer: The most common source of this issue is an incomplete resuspension of your gBlocks Gene Fragment. The pellets can stick to the side or cap of the tube, and unless you vortex vigorously and incubate the tube at 50°C for 20 minutes, you might not have the DNA concentration you expect. We recommend the following:

1. Vortex your tube, and incubate at 50°C for 20 minutes.
2. Vortex vigorously again and spin the tube down.
3. Recheck the concentration of your solution. (In general, it is always a good idea to recheck your concentration after you resuspend a DNA sample.)

Question: I found a mutation in my clone. Is there a problem with the gBlocks Gene Fragment?

Answer: gBlocks fragments are not clonal products and are synthesized using both chemical and enzymatic steps, so there will be a subpopulation in the tube with truncations, deletions, or mutations. Fortunately, the large majority of sequences should be correct, so by sequencing a few more colonies you should find a correct clone.