DNA synthesis and transformation
All designed DNA sequences will be chemically synthesized and subclone into a gateway cloning vector, pMDC32 (Figure 1). pMDC32 vector has double 35S constitutive promoters. So the designed sequence will be always highly expressed. Using this cloning vector, we will transform your designed DNA sequence into Arabidopsis. GenoCon 2010 used next generation of T2 seed for the following assay.
Figure 1. Gateway cloning vectore, pMDC32.
- Link to the description how the pMDC32 vector works.
- Link to the explanation of transformation; — how your designed DNA sequences will be experimentally introduced into Arabidopsis thaliana.
Formaldehyde resistance assay
Antibiotic selection
After the transformation of your designed sequence into Arabidosis, we will use antibiotics of hygromycin to select successfully transformed seedlings. Arabidopsis seeds will be plated on 1% agar containing MS medium and hygromycin. The seedlings will be incubated for 8 days at 22°C in 24 hr continuous light. Then we will transplant the seedlings on antibiotics free media and incubate for 16 days under 24 hr continuous light.
Formaldehyde treatment
The developed plants will be transplanted into several density of formaldehyde-containing media and incubated in 24 hr continuous light and at 22℃.
Image analysis
After transplanting on formaldehyde-containing media, we will take pictures for 14 days. Image analysis is explained on GenoCon 2010 web site Image analysis.
