Steven Ghivizzani (University of Florida, Gainesville, FL, USA) described a novel approach to enhancing cartilage repair, in which bone marrow is aspirated and, as it clots, mixed with vectors carrying chondrogenic genes.
To obtain transgenic 10UAS-Cas9 flies, the PiggyBac-10UAS-cas9 vector was mixed with helper vector PiggyBac-transposase (Thibault et al. 2004) and injected into w fly embryos.
To obtain transgenic vasa -Cas9 flies, the PiggyBac- vasa -cas9 vector was mixed with helper vector PiggyBac-transposase (Thibault et al. 2004) and injected into w fly embryos.
The second most prevalent failure mode in the "process related" category, "Mixed clone with vector sequence" (30% of reads in this classification), is likely a result of two side by side colonies being picked into the same well or through contamination originating from the use of automated liquid handlers.
The nematodes treated with primary and secondary antibodies were mixed with Vectashield (Vector, Burlingame, CA) and mounted on glass slides.
After the second round of PCR the products are mixed with the vector, treated with UDG and allowed to anneal.
The annealed oligos were kinased, and finally mixed with the vector in a ligation reaction, producing pTSH117, with 10 bp within the fim and hin mirrored inverted repeats, and pTSH118, with 30 bp within the fim mirrored inverted repeats and 10 bp within the hin mirrored repeat.
PCR products were treated with T4 polymerase, mixed with linearized vector (digested with SmaI and treated with T4 polymerase) and incubated for 15 min at room temperature.
75 to 200 ng of expression plasmid (depending on construct) was mixed with empty vector to a total of 700 ng DNA in 16μl PBS.
