Efforts to transform Heterosigma akashiwo using an Agrobacterium-mediated approach v3 (protocols.io.4ysgxwe) [dataset]

Vinay K, Monica Accerbi, Pamela J
2019 protocols.io  
1 1 1 1 This protocol was developed for transformation of Heterosigma akashiwo by Agrobacterium tumefaciens. H. akashiwo does not grow on solid medium, so transformants are grown "in bulk" and should not be considered clonal. We have had limited success with this protocol and may require further optimization. 1 Plant transformation binary vectors (here pCambia1300 and its variants) were introduced into Agrobacterium tumefaciens (GV3101(pMP90)) via electroporation. Cells were grown at 29 C for 2
more » ... grown at 29 C for 2 days on agar-solidified LB (Luria-Bertani) medium with antibiotics (Kanamycin 50 μg/ml; Rifampicin 25 μg/ml; Gentamycin 25 μg/ml). Positive colonies carrying the plasmid were identified using PCR. 2 Agrobacterium starter cultures (5 ml LB broth) with appropriate antibiotics were grown overnight at 29 C at 200 rpms. Approximately 16-18 h prior to the algal transformation a larger culture (25 ml) was inoculated with 25 μl of the starter culture The following day, Agrobacterium cultures which had reached an OD 600 of 1.5 were pelleted at 9,000 rpm for 5 min.
doi:10.17504/protocols.io.4ysgxwe fatcat:qgdnejesdve6xhy5i5s6lbbu6m