DNA Assembly Webinar Questions and Answers

Your colleagues submitted interesting questions during the webinar, “Move beyond traditional DNA cloning and editing,” so we’ve collected and answered them here.

Find the answer quickly by clicking your desired question.

Q- What if my fragments contain repetitive sequences?

Q- Can I clone 5 fragments using the seamless assembly kit, even if it’s not supported by your web tool? 

Q- What tips can you provide to minimize the appearance of “empty vector” clones?

Q-What if my fragments contain repetitive sequences? 

A- Since most commercially available seamless assembly kits, including our GeneArt® Seamless kits, are based on homologous recombination, cloning and assembly of fragments with repetitive regions could result in rearrangements in your final construct.  Life Technologies will have a cloning and assembly kit for repetitive sequences in 2013.

Q- Can I clone 5 fragments using the seamless assembly kit, even if it’s not supported by your web tool?

A- Yes, absolutely.  Primers for incorporating end-homology can be designed ‘by hand’ with rules provided in the kit manuals.  If the final size of the construct is much larger than kit recommendations, we recommend you screen more colonies than the manual suggests to ensure isolating the correct clone.

Q- What tips can you provide to minimize the appearance of “empty vector” clones?

A- Cut the cloning vector with 2 different restriction enzymes to make non-compatible ends.