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.
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:
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.
This is the tagline
This is a normal body paragraph. The available space here will depend on your form height: you may need to be parsimonius with shorter forms, and if your form is very long, you'll probably want to add an image and/or additional content.
This is the title/intro for an unordered list