The Ti vector for transforming plants via agro (pPZP, pGreen, pClean, etc) is about 10k in length. The Ti region that agro cuts and packs hasn't really been put through the ringer in terms of seeing how much it can incorporate. I've worked with 10kbp+ and it did fine. Transformation via agro is simple. Personally I hate Arabi floral dip.
I just grow agro with plasmid to OD600 = 0.5, incubate leaf disks of month old plantlets (1cm dia.) in 15mL of liquid culture, then dab on sterile cotton pad or gauze, and plate on MS BAP NAA and Agar pH 5.8 (tobacco) for 48hrs or until decent bacterial halo forms around explant. Then transfer to MS BAP NAA Agar and selection herbicide (I like kanamycin 50-100mg/L) and let nature do the rest.
Replate every two weeks till shoots form, transfer shoots to rooting media (MS NAA Agar Kana) and wait till roots form. If strong roots form and dig into media then u have transformants (verify by pcr to be sure). Transfer to soil and slowly lower humidity. Cool white lights all the way through. 100 microeinsteins or more. 16hr days. Works for me every time.
Have done well over 700 transformations and less than 0.1% were false positives. Results may vary based on experience but tobacco is easy and not as sensitive to environs as Arabidopsis. There is no rational reason why I hate floral dip. Arabi seeds are terrible to handle, Silwet is expensive, and overall a boring experience.
Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D
I just grow agro with plasmid to OD600 = 0.5, incubate leaf disks of month old plantlets (1cm dia.) in 15mL of liquid culture, then dab on sterile cotton pad or gauze, and plate on MS BAP NAA and Agar pH 5.8 (tobacco) for 48hrs or until decent bacterial halo forms around explant. Then transfer to MS BAP NAA Agar and selection herbicide (I like kanamycin 50-100mg/L) and let nature do the rest.
Replate every two weeks till shoots form, transfer shoots to rooting media (MS NAA Agar Kana) and wait till roots form. If strong roots form and dig into media then u have transformants (verify by pcr to be sure). Transfer to soil and slowly lower humidity. Cool white lights all the way through. 100 microeinsteins or more. 16hr days. Works for me every time.
Have done well over 700 transformations and less than 0.1% were false positives. Results may vary based on experience but tobacco is easy and not as sensitive to environs as Arabidopsis. There is no rational reason why I hate floral dip. Arabi seeds are terrible to handle, Silwet is expensive, and overall a boring experience.
Sebastian S. Cocioba
CEO & Founder
New York Botanics, LLC
Plant Biotech R&D
From: Yuriy Fazylov
Sent: 7/22/2014 5:25 PM
To: diybio@googlegroups.com
Subject: [DIYbio] Re: Yuriy's fundraiser on experiment.com
0 comments:
Post a Comment